Protein Research Workflows: From Expression to Analysis

Protein research handbook Cloning and protein expression • Protein sample preparation • Protein purification • Gel electrophoresis • Western blotting • Immunoassays • Mass spectrometry • Modification and crosslinking • Cryo-EM sample preparation Protein biology Detect, measure, and analyze protein expression, identity, and function using our portfolio of protein biology products. Protein biology encompasses both the study of the structure and function of proteins as the primary focus of investigation and the use of antibodies, proteins, and peptides as tools to purify, detect, and characterize biological systems. Certain methods of protein analysis, such as immunoprecipitation, western blotting, and ELISA, have been used routinely in laboratories for many years. Others, such as quantitative protein mass spectrometry and cryo-EM, are relatively recent and rapidly developing technologies. New tools and products are continually being developed to advance all aspects of protein biology research. Cloning and protein expression 5 Protein sample preparation 12 Protein purification 29 Protein gel electrophoresis 41 Western blotting 57 Immunoassays 73 Mass spectrometry 87 Protein bioconjugation, crosslinking, and modification 101 Cryo-EM sample preparation 112 Resources 119 Contents Cloning and protein expression Recombinant protein expression technology enables analysis of gene regulation and protein structure and function. Utilization of recombinant protein expression varies widely—from investigation of function in vivo to large-scale production for structural studies and biotherapeutic drug discovery. Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Tools to optimize your cloning step 7 Recombinant protein expression systems 8 Ordering information 11 Cloning and protein expression thermofisher.com/proteinbiology 5 Introduction To investigate how particular proteins regulate biology, researchers usually require a means of producing (manufacturing) functional proteins of interest. Given the size and complexity of proteins, de novo synthesis is not a viable option for this endeavor. Instead, living cells or their cellular machinery can be harnessed as factories to build and construct proteins based on supplied genetic templates (Figure 1). Most vectors contain a promoter for expression by a specific host system; however, some offer the option to add your own promoter. Once cloning is completed, plasmids are taken up into Using the right expression system for your specific application is the key to success. Protein solubility, functionality, purification speed, and yield are often crucial factors to consider when choosing an expression system. Additionally, each system has its own strengths and challenges, which are important when choosing an expression system. For example, bacterial host cells are low-cost, easy to culture, and easily scalable but are limited to the expression of bacterial proteins or simple eukaryotic proteins with limited posttranslational modifications. Since most proteins undergo some degree of posttranslational modification, bacterial host cells limit the range of the proteins expressed. Insect and mammalian host systems support the most complex proteins and maximum protein quality. Insect host systems support multi-protein complexes with posttranslational modifications similar to mammalian cells and are a good option for proteins that are toxic to mammalian cells. If the protein studied must be identical to its in vivo mammalian counterpart, then a mammalian host system is ideal. Mammalian host systems retain the most posttranslational modifications and most closely resemble functional human proteins. However, these systems are also the most expensive. Both insect and mammalian cell lines have more demanding culture conditions, and therefore are not suited to every protein expression case. The most common mammalian cell lines are human embryonic kidney (HEK293) cells and Chinese hamster ovary (CHO) cells. Choice of cell line may also depend on whether the protein of interest expresses better in one cell line or the other, or whether the researcher wants to take a transient or stable expression strategy. Transient expression usually implies a short-term, small-scale production, while stable expression involves the long-term integration of genes into the genome for large-scale production. However, new technologies have transformed transient expression to have robust yields. In prokaryotes, the processes of transcription and translation occur simultaneously. In eukaryotes, the processes are spatially separated and occur sequentially—with transcription happening in the nucleus and translation occurring in the cytoplasm. After translation, polypeptides are modified in various ways to complete their structure, designate their location, or regulate their activity within the cell. Posttranslational modifications (PTMs) are various additions or alterations to the chemical structure of the newly synthesized protein and are critical features of overall cell biology. Cloning refers to the propagation of DNA of interest from an existing organism. This can be accomplished by cloning a gene of interest into an expression vector (Figure 2). DNA AAAAA mRNA Protein Transcription Translation Figure 1. Transcription and translation. Information is flowed from DNA base-pair sequence (gene) to amino acid polypeptide sequence (protein). Figure 2. Example of an expression vector. Choice of promoter: • CMV • EF-1 • UbC • T7 or SP6 • Other Marker for propagation in E. coli Optional epitope tags: • V5 • Myc • His • Xpress • Other Choice of cloning method: • Restriction cloning • Gibson Assembly cloning Origin of replication examples: • pUC ori • pBR322 ori • Gateway technology • TOPO cloning Additional antibiotic selection marker: • Neomycin (Geneticin antibiotic) • Blasticidin S • Hygromycin B • Zeocin antibiotic • Other Expression vector map GOI Ampicillin ri Promoter Selection marker O Table 1. Overview of epitope tags. Purpose Description Examples of tags Detect Well-characterized antibody available against the tag; easily visualized V5, Xpress, Myc, 6xHis, GST, BioEase, capTEV, GFP, Lumio, HA, FLAG tag Purify Resins available to facilitate purification 6xHis, GST, BioEase, capTEV tag Cleave Protease recognition site (TEV, EK, HRV 3C, factor Xa) to remove tag after expression to obtain native protein Any tag with a protease recognition site following the tag (only on N terminus) competent cells (e.g., chemically competent or electrocompetent E. coli) for propagation and storage, by a process called transformation. Epitope tags can be used to allow for easy detection or rapid purification of your protein of interest by fusing a sequence coding for the tag to your gene (Table 1). 6 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiology Tools to optimize your cloning step GeneArt Gene Synthesis and GeneOptimizer software The power of custom gene synthesis is the ability to design your DNA without the constraints of traditional cloning. Equally important to most researchers, however, is obtaining high yields of mRNA and, ultimately, protein from synthetic genes. We developed Invitrogen™ GeneArt™ GeneOptimizer™ software to maximize the expression of synthetic genes in all commonly used expression systems. Tuning the expression level by choosing the optimal promoter and terminator combination can be an essential part of your expression project. The origin of replication also has a significant influence on the expression level of the foreign protein. We offer a broad range of commercially available, predesigned vectors optimized for various expression systems. Learn more at thermofisher.com/genesynthesis Gibson Assembly kits and GeneArt Strings DNA Fragments Invitrogen™ GeneArt™ Gibson Assembly® cloning kits provide highly efficient, seamless cloning, enabling the assembly of multiple DNA fragments of varying length into any vector. When combined with Invitrogen™ GeneArt™ Strings DNA Fragments or Invitrogen™ GeneArt™ Gene Synthesis, these cloning kits can be used to build simple constructs as well as large and demanding constructs from multiple fragments. Learn more about GeneArt Gibson Assembly cloning kits for protein expression at thermofisher.com/gibsonassembly Gateway cloning Invitrogen™ Gateway™ cloning technology is rapid, robust, and suited for high-throughput clone generation for protein production. It allows shuffling between different expression systems in just a few simple steps. Choose from a diverse selection of host systems, including E. coli, yeasts, and insect or mammalian cells, each of which utilizes unique destination vectors for all your expression applications. Additionally, the Invitrogen™ Gateway™ Vector Conversion System can convert specialized or customized vectors into an Invitrogen™ Gateway™ destination vector to suit your expression workflow. Select the right Gateway vectors for your workflow and review our cloning protocols at thermofisher.com/gateway One Shot chemically competent E. coli E. coli cells are widely used for production of recombinant proteins quickly, economically, and on a large scale. The most popular strain for recombinant protein expression is BL21 and its derivatives. Invitrogen™ One Shot™ chemically competent cells, such as BL21 Star™ (DE3) and BL21-AI™ cells, are optimized for high-level protein or toxic protein expression from T7 promoters. All are induced with IPTG or L-arabinose and come in the convenient Invitrogen™ One Shot™ format, allowing for transformation and recovery in a single tube. Choose chemically competent cells optimized for protein expression at thermofisher.com/compcells Cloning and protein expression thermofisher.com/proteinbiology 7 Recombinant protein expression systems Achieving optimal and reliable amounts of recombinant protein is easier because of our wide selection of trusted mammalian, insect, yeast, bacterial, and cell-free protein expression systems (Table 2). Backed by a team of experienced professionals to help you quickly optimize your protein expression experiments, we can accelerate your research and development by offering: • Higher protein yields (3–20x higher than other systems) • Faster protein production (days vs. weeks or months) • High cell density • Lower cost per mg of protein • Complete, optimized systems of cells, media, transfection reagents, enhancers, feeds, and vectors Table 2. Expression system selection guide. Expression system Human (HEK293) Hamster (CHO) Featured application Membrane proteins (GPCRs, ion channels, other membrane proteins) Ig-related proteins Other applications • Difficult-to-express proteins • Secreted proteins • Membrane proteins • Secreted proteins Posttranslational modifications Full Nearly full Protein yield Up to 1 g/L Up to 3 g/L Recommended host system kit Expi293 expression system kits ExpiCHO Expression System Kit Recommended cells Expi293F Cells Expi293F GnTI– Cells Expi293F Inducible Cells Expi293F Inducible GnTI– Cells ExpiCHO-S Cells (cGMP Banked) ExpiCHO-S Cells Recommended media Expi293 Expression Medium ExpiCHO Expression Medium Recommended transfection and delivery ExpiFectamine 293 Transfection Kit ExpiFectamine CHO Transfection Kit Recommended expression vectors pcDNA3.4 TOPO vector pcDNA3.4 TOPO vector Recommended cultureware Nalgene shake flasks Nunc bioreactor tubes Nunc plates Nalgene shake flasks Nunc bioreactor tubes Nunc plates Recommended extraction reagent kit M-PER Mammalian Protein Extraction Reagent M-PER Mammalian Protein Extraction Reagent Recommended protein labeling Expi293 Met (–) Protein Labeling Kit L-Methionine (Methyl-13C) L-Selenomethionine Scale-up Expi293 Expression Medium Single-use bioreactor vessels ExpiCHO Stable Production Medium EfficientFeed C+ 2X Supplement Efficient-Pro Feed 2 Single-use bioreactor vessels Services option Gene-to-protein services Gene-to-protein services The Gibco™ Expi™ platforms are examples of expression systems that are fast and cost-effective with high yield. These systems combine the advantages of both transient and stable strategies for high expression of diverse proteins (Figure 3). For example, the Gibco™ Expi293™ Expression System uses HEK293 cells and can yield up to 1 g/L of protein in just 5–7 days. Figure 3. External collaborator results. In collaboration with 22 labs, expression levels of 98 different proteins were tested using the Expi293 Expression System. The following results were obtained: • 87% of proteins demonstrated increased expression in the Expi293 system compared with the user’s current system • 4.6x average increase for all proteins (n = 98) • 4.0x average increase for mAbs (n = 54); highest level was 826 mg/L • 5.3x average increase for non-mAbs (n = 44); highest level was 790 mg/L 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Fold yield increase 8 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiology Insect (Sf9, Sf21) Yeast Bacterial Cell-free Intracellular proteins Industrial enzymes Bacterial proteins Rapid protein expression • Toxic proteins • Multi-protein complexes Low-complexity proteins Low-complexity proteins • Toxic proteins • In vitro labeling Partial Partial Limited Partial Up to 900 mg/L Up to 10 g/L Up to 10 mg/L Up to 750 mg/L ExpiSf Expression System Starter Kit EasySelect Pichia Expression Kit Champion pET SUMO Expression System pBAD TOPO TA Expression Kit Cell-free in vitro ExpiSf9 Cells PichiaPink Expression Strain Set GS115, Pichia pastoris Yeast Strain X-33, Pichia pastoris Yeast Strain One Shot BL21 Star (DE3) Chemically Competent E. coli cells BL21-AI One Shot Chemically Competent E. coli cells HeLa and CHO extracts ExpiSf CD Medium PichiaPink Media Kit YPD Broth MagicMedia E. coli Expression Medium LB Broth None required ExpiFectamine Sf Transfection Reagent Neon Transfection System Starter Pack None required None required pFastBac 1 vector pFastBac Dual Expression Vector pPINK (LC and HC) pPICZα A, B, and C Pichia Vectors pPICZ A, B, and C Pichia Vectors Champion pET SUMO vector pBAD TOPO vector pRSET bacterial expression vectors T7 cell-free expression vectors Nalgene shake flasks Nunc bioreactor tubes Nunc plates Nalgene shake flasks Nalgene shake flasks I-PER Insect Cell Protein Extraction Reagent Y-PER Yeast Protein Extraction Reagent B-PER Complete Bacterial Protein Extraction Reagent None required ExpiSf CD Medium Single-use bioreactor vessels Bacto CD Supreme Fermentation Production Medium (FPM) Single-use bioreactor vessels Gene-to-protein services Cloning and protein expression thermofisher.com/proteinbiology 9 Gibco™ ExpiCHO™ Expression System • Maintain protein quality and function and easily transition from research to production using the Gibco™ ExpiCHO-S™ cGMP-banked cell line and Gibco™ ExpiCHO™ Stable Production Medium • Get the highest yields possible in a transient expression system (up to 3 g/L). Learn more at thermofisher.com/expicho Gibco™ Expi293™ Expression System Kit • Rapid production of protein in just 5–7 days • Specialized cell lines for structural biology research, such as Gibco™ Expi293F™ cell lines, are available Learn more at thermofisher.com/expi293 Gibco™ ExpiSf™ Expression System • First chemically defined baculovirus expression system • Generate 3x more protein compared to existing insect expression platforms Learn more at thermofisher.com/expisf Test your protein research knowledge Question: Which of the following are some of the most common mammalian cell lines used for foreign protein expression? (Select all that apply.) A. HeLa B. CHO C. Jurkat D. HEK293 Answer: B and D 10 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiology Ordering information Product Quantity Cat. No. Mammalian cell–based systems Expi293 Expression System Kit 1 kit A14635 Expi293 Expression Medium 1,000 mL A1435101 ExpiFectamine 293 Transfection Kit 1 kit A14524 Expi293 GnTI– Expression System Kit 1 kit A39250 ExpiCHO Expression System Kit 1 kit A29133 ExpiCHO Expression Medium 1,000 mL A2910001 ExpiFectamine CHO Transfection Kit 1 kit A29129 To view additional products, go to thermofisher.com/mammalianexpression Insect cell–based systems ExpiSf Expression System Starter Kit 1 kit A38841 ExpiSf CD Medium 500 mL A3767801 ExpiFectamine Sf Transfection Reagent 1 mL A38915 To view additional products, go to thermofisher.com/insectexpression Yeast cell–based systems PichiaPink Secretion Optimization Kit 1 kit A11150 EasySelect Pichia Expression Kit 1 kit K174001 Pichia Expression Kit, original kit 1 kit K171001 To view additional products, go to thermofisher.com/yeastexpression Bacterial cell–based systems GeneArt Gibson Assembly HiFi Master Mix 50 reactions A46628 Gateway LR Clonase II Enzyme Mix 20 reactions 11791020 Champion pET100 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot Chemically Competent E. coli 20 reactions K10001 pBAD202 Directional TOPO Expression Kit 20 reactions K420201 One Shot BL21 Star (DE3) Chemically Competent E. coli 20 reactions C601003 BL21-AI One Shot Chemically Competent E. coli 20 reactions C607003 MagicMedia E. coli Expression Medium 1 L K6803 To view additional products, go to thermofisher.com/bacterialexpression Cell-free systems 1-Step Human Coupled IVT Kit – DNA 40 reactions (25 μL each) 88882 1-Step Human High-Yield Mini IVT Kit 10 reactions (100 μL each) 88891 1-Step Human High-Yield Maxi IVT Kit 2 reactions (2 mL each) 88892 Retic Lysate IVT Kit 60 reactions AM1200 To view additional products, go to thermofisher.com/cellfreeexpression For more information, or to view additional pack sizes, expression vectors, media, and cell lines, go to thermofisher.com/proteinexpression Cloning and protein expression thermofisher.com/proteinbiology 11 Protein sample preparation Because of the heterogeneity of proteins, there is no one method or reagent that is optimal for general protein isolation and purification. The first step in protein analysis is cellular extraction. Following lysis, and depending on the next step in the workflow, the protein extract may require further cleanup, using techniques such as dialysis, desalting, or concentration. After any cleanup procedure, the amount of protein in the sample should be determined using any one of numerous protein assays. Protein extraction reagents and kits 14 Detergents 16 Protease and phosphatase inhibitors 17 Slide-A-Lyzer dialysis products 18 Zeba desalting products 20 Protein concentrators 22 Protein quantitation products 23 Ordering information 26 Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting 12 Protein sample preparation thermofisher.com/proteinbiology Protein extraction Protein extraction techniques vary depending on the source of the starting material, the location within the cell of the protein of interest, and the downstream application. Historically, mechanical disruption has been used to lyse cells and tissues, but detergentbased solutions have more recently been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical methods. However, cell lysis disrupts cell membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent these negative effects, protease and phosphatase inhibitors can be added to the lysis reagents. Numerous compounds have been identified and used to inactivate or block the activities of proteases and phosphatases by reversibly or irreversibly binding to them. Protein cleanup Many detergents and salts used in protein extraction formulations may have adverse effects on protein function or stability, or may interfere with downstream analysis; therefore, it may be necessary to remove or reduce these contaminants following cell lysis, using techniques such as dialysis or desalting. In addition, if the protein sample is too dilute, it may require concentration. Dialysis facilitates the removal of small unwanted compounds from protein in solution by selective diffusion through a semipermeable membrane. Proteins that are larger than the membrane pores are retained on the side of the membrane, but small contaminants diffuse freely through the membrane and approach an equilibrium concentration. Desalting, also known as size exclusion chromatography or gel filtration, utilizes a resin with pores that are large enough for small contaminants (e.g., salts) to penetrate, but too small for the protein of interest to enter. This causes the contaminants to slow down their rate of migration, and the larger, faster proteins separate from the slower, smaller molecules. Protein concentration and diafiltration use a semipermeable membrane to separate macromolecules from low molecular weight compounds via centrifugation. During concentration, the low molecular weight solutes are forced through the membrane while the macromolecules remain on the sample side of the membrane, where they become concentrated to a smaller volume as the reagent is forced across the membrane to the opposite side. Protein assays Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration. Colorimetric, reagent-based protein assay techniques have been developed that are used by nearly every laboratory involved in protein research. Protein is added to the reagent, producing a color change in proportion to the amount added. Protein concentration is determined by reference to a standard curve consisting of known concentrations of a purified reference protein. Unfortunately, no protein assay method exists that is either perfectly specific to proteins or uniformly sensitive to all protein types. Therefore, successful use of protein assays involves selecting the method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain. Protein sample preparation thermofisher.com/proteinbiology 13 Protein extraction reagents and kits Gentle formulations designed to maximize protein yield and activity Obtain high protein yield from tissues, cells, or subcellular fractions using reagents and kits that are optimized for mammalian, bacterial, yeast, insect (baculovirus), and plant samples (Table 1). These gentle formulations have been verified in multiple tissue types and cell lines, and generally eliminate the need for mechanical cell disruption. These extracts are compatible with a wide range of downstream applications, including protein assays, immunoprecipitation, protein purification, immunoassays, western blotting, gel shift assays, and enzyme assays. Table 1. Overview of sample types and protein extraction reagents and kits. Sample type Goal Recommended Thermo Scientific™ reagents or kits Primary or cultured mammalian cells or tissues • Total protein extraction • M-PER reagent • T-PER reagent • N-PER reagent • RIPA Lysis and Extraction Buffer • Pierce IP Lysis Buffer Cultured mammalian cells or tissues • Subcellular fractionation or organelle isolation • NE-PER reagent • Subcellular Fractionation Kits • Mitochondria Isolation Kits • Pierce Cell Surface Protein Isolation Kit • Syn-PER Reagent • Lysosome Enrichment Kit Bacterial cells • Total protein extraction • B-PER reagent Yeast cells • Total protein extraction • Y-PER reagent Insect cells (baculovirus) • Total protein extraction • I-PER reagent Plant tissue (leaf, stem, root, flower) • Total protein extraction • P-PER reagent 14 Protein sample preparation thermofisher.com/proteinbiology Membrane Cytosolic Total 0 200 400 600 800 1,000 1,200 1,400 Protein yield (μg) HeLa cells (5 x 106) 0 500 1,000 1,500 2,000 2,500 3,000 Membrane Cytosolic Total Protein yield (μg) Liver tissue (30 mg) Mem-PER Plus kit Mem-PER kit (original) EMD Millipore ProteoExtract kit Bio-Rad ReadyPrep kit Membrane Cytosolic Total 0 200 400 600 800 1,000 1,200 1,400 Protein yield (μg) HeLa cells (5 x 106) 0 500 1,000 1,500 2,000 2,500 3,000 Membrane Cytosolic Total Protein yield (μg) Liver tissue (30 mg) Mem-PER Plus kit Mem-PER kit (original) EMD Millipore ProteoExtract kit Bio-Rad ReadyPrep kit Figure 1. Improved protein yield using the Thermo Scientific™ Mem-PER™ Plus Membrane Protein Extraction Kit. Membrane proteins were isolated from mouse liver tissue and HeLa cells using four commercial extraction kits. Protein yields for membrane, cytosolic, and total fractions were determined using the Thermo Scientific™ Pierce™ BCA Protein Assay Kit. A B Figure 2. Nuclear and cytosolic fractions are obtained with minimal cross-contamination. HeLa cell proteins were extracted with Thermo Scientific™ NE-PER™ Nuclear and Cytoplasmic Extraction Reagents or with nuclear extraction kits from other vendors. Samples of the nuclear and cytosolic fractions were analyzed by western blot using antibodies against common nuclear and cytoplasmic protein markers and visualized using Thermo Scientific™ SuperSignal™ West Pico Chemiluminescent Substrate. Nuclear fractions produced with the NE-PER kit had minimal to no contamination with cytosolic proteins. Thermo Scientific C N C N C N C N Sigma Active Motif Millipore Thermo Scientific C N C N C N C N Sigma Active Motif Millipore Cytosolic Nuclear HSP90 MAPK Calcineurin SP1 CREB HDAC1 For more information, or to view additional products, go to thermofisher.com/proteinextraction Highlights: • Optimized—formulations maximize protein yield and preserve protein activity (Figure 1) • Efficient—only produces minimal cross-contamination between subcellular fractions (Figure 2) • Compatible—extracts can be used directly in most downstream applications • Gentle—eliminates the need for mechanical cell disruption for most sample types Protein sample preparation thermofisher.com/proteinbiology 15 Detergents Easy-to-pipette, highly purified Surfact-Amps 10% solutions Thermo Scientific™ Surfact-Amps™ Detergent Solutions are easy-to-use 10% (w/v) solutions of highly purified detergents that can be used in routine and high-demand protein research methods and molecular biology techniques (Figure 3). These formulations provide high purity, quality, and stability. Unlike neat detergents, which are extremely viscous, Surfact-Amps 10% solutions are easy to pipet accurately. The surfactant solutions are carefully prepared and packaged under nitrogen in glass ampules or non-leaching HDPE bottles, helping to ensure their stability and minimizing the accumulation of peroxides and degradation products. Highlights: • Accurate—precise 10% detergent solution in ultrapure water • Easy to use—solution is simple to dispense and dilute • Exceptionally pure—less than 1 μeq/mL peroxides and carbonyls (Tables 2 and 3) • Stable—packaged under inert nitrogen gas in glass ampules or HDPE bottles Hydrophobic tail Hydrophilic head Figure 3. General structure of a detergent molecule. Table 3. Purity comparison of NP-40 detergents. Manufacturer/brand Peroxide concentration (μeq/mL) Carbonyl concentration (μeq/mL) Thermo Scientific ≤0.035 ≤0.01 Amresco 0.083 0.374 Anatrace 0.053 4.246 G-Biosciences ≤0.035 ≤0.01 Millipore ≤0.035 0.042 Roche 0.056 0.021 Peroxide levels were measured using Thermo Scientific™ Pierce™ Quantitative Peroxide Assay Kit, and carbonyl levels were measured using the Brady test for carbonyls. Table 2. Purity comparison of Tween 20 detergents. Manufacturer/brand Peroxide concentration (μeq/mL) Carbonyl concentration (μeq/mL) Thermo Scientific ≤0.01 ≤0.32 Amresco 0.598 0.399 Anatrace ≤0.01 ≤0.32 G-Biosciences 0.718 ≤0.32 Millipore 0.037 ≤0.32 Roche 0.279 0.445 Peroxide levels were measured using Thermo Scientific™ Pierce™ Quantitative Peroxide Assay Kit, and carbonyl levels were measured using the Brady test for carbonyls. For more information, or to view additional products, go to thermofisher.com/detergents 16 Protein sample preparation thermofisher.com/proteinbiology Protease and phosphatase inhibitors Broad-spectrum liquid cocktails and tablets for complete protein protection Protease and phosphatase inhibitor cocktails and tablets are ideal for the protection of proteins during extraction or lysate preparation from primary cells, cultured mammalian cells, animal tissues, plant tissues, yeast cells, or bacterial cells. Formulations are packaged in multiple sizes, and EDTA-free versions are available for assays that are sensitive to divalent cations. Highlights: • Convenient—ready-to-use, fully disclosed, broad-spectrum formulations available as either liquid cocktails or tablets in multiple pack sizes and with a minimum one year shelf life • Complete protection—combined cocktail available with all-in-one formulations containing both protease and phosphatase inhibitors (Table 4) • Compatible—use directly with Thermo Scientific™ Pierce™ cell lysis buffers or other commercial or homemade detergent-based lysis reagents Table 4. Components present in Thermo Scientific™ Halt™ Inhibitor Cocktails and Thermo Scientific™ Pierce™ Protease and Phosphatase Inhibitor Tablets. Inhibitor component Target (mechanism) Protease liquid cocktails and tablets Phosphatase liquid cocktails and tablets Combined protease and phosphatase liquid cocktails and tablets AEBSF•HCl Serine proteases (irreversible) • Aprotinin Serine protease (reversible) • • Bestatin Aminopeptidase (reversible) • • E-64 Cysteine (irreversible) • • Leupeptin Serine and cysteine proteases (reversible) • • Pepstatin Aspartic acid proteases (reversible) • EDTA* Metalloproteases (reversible) • • Sodium fluoride Serine/threonine and acidic phosphatases • • Sodium orthovanadate Tyrosine and alkaline phosphatases • • β-Glycerophosphate Serine/threonine phosphatase • • Sodium pyrophosphate Serine/threonine phosphatase • • * EDTA is not in EDTA-free formulations. For more information, or to view additional products, go to thermofisher.com/inhibitorcocktails Protein sample preparation thermofisher.com/proteinbiology 17 Slide-A-Lyzer dialysis products Easy-to-handle devices, cassettes, and flasks for secure sample processing Thermo Scientific™ dialysis units facilitate rapid and trouble-free dialysis of sample volumes from 10 μL to 250 mL. Unlike standard flat tubing, these innovative devices do not require knots or clips that can lead to leaking and sample loss. Thermo Scientific™ Pierce™ 96-Well Microdialysis Plates and Slide-A-Lyzer™ MINI Dialysis Devices are ideal for small volumes, Slide-A-Lyzer™ G3 Dialysis Cassettes are recommended for small to medium volumes, and Slide-A-Lyzer™ Dialysis Flasks are recommended for larger volumes (Table 5). Highlights: • Excellent sample recovery—low-binding plastics and membranes help minimize sample loss compared to filtration and resin systems, while maximizing dialysis speed (Figures 4 and 5) • Convenient—easy-to-grip design helps simplify sample addition and removal with syringe or pipette • Secure—sealed membranes help prevent leakage that can occur with dialysis tubing and homemade devices • Validated—each device is leak-tested during production Table 5. High-performance dialysis product selection guide. 10–1,000 μL 10–2,000 μL 1–125 mL 150–250 mL 15–100 mL Membrane MWCO Pierce Microdialysis Plate Slide-A-Lyzer MINI Dialysis Device Slide-A-Lyzer G3 Dialysis Cassette Slide-A-Lyzer Dialysis Flask Thermo Scientific™ SnakeSkin™ Dialysis Tubing 2K • • • • NA 3.5K • • • • • 7K NA • NA NA • 10K • • • • • 20K • • • • NA Figure 4. Sample retention by the Thermo Scientific™ Slide-A-Lyzer™ cassette membranes with 2K, 3.5K, 7K, 10K, or 20K molecular weight cutoff (MWCO). Individual proteins or vitamin B12 (1 mg/mL) in either saline or 0.2 M carbonate-bicarbonate buffer, pH 9.4, were dialyzed overnight (17 hr) at 4°C. The amount of retentate was estimated using either the Pierce BCA Protein Assay Kit or absorption at 360 nm (for vitamin B12). 0 40 60 80 70 50 30 10 90 100 20 % Retention 0 40 60 80 70 50 30 10 90 100 20 % Retention 0 40 60 80 70 50 30 10 90 100 20 % Retention 0 40 60 80 70 50 30 10 90 100 20 % Retention 0 40 60 80 70 50 30 10 90 100 20 % Retention 2K MWCO 3.5K MWCO 7K MWCO 10K MWCO 20K MWCO Vitamin B12 (1.4K MW) Bacitracin (1.4K MW) Tyrosine protein kinase (1.7K MW) Biotin-TPK (1.9K MW) Protein kinase Ce peptide (2K MW) Insulin A chain (2.5K MW) Insulin B chain (3.5K MW) Insulin (5K MW) Cytochrome c (12.4K MW) Lysozyme (14K MW) Myoglobin (17K MW) 18 Protein sample preparation thermofisher.com/proteinbiology Figure 5. The rate of removal of NaCl using various dialysis products. NaCl removal from samples was determined by measuring the conductivity of the retentate at the indicated times. (A) Slide-A-Lyzer MINI Dialysis Device (10K MWCO, 2 mL) vs. conventional dialysis. Bovine serum albumin (BSA) samples (2 mL, 0.25 mg/mL in 1 M NaCl) were dialyzed against 45 mL of water in 50 mL disposable conical tubes on an orbital shaker (300 rpm) at room temperature. The water was changed once after 2 hr. Results are the average of two samples. For conventional dialysis, the samples were dialyzed against 2 L of water in a beaker with stirring. More than 95% of NaCl was removed within 4 hr. (B) Samples of 0.1 mL of 0.4 mg/mL cytochrome c in 1 M NaCl were dialyzed in the Pierce 96-Well Microdialysis Plate against 1.8 mL of water at room temperature with gentle shaking. The buffer was changed hourly over a 4 hr period. Removal of NaCl was >83% after 2 hr and >99% after 4 hr. (C) Proteins in 200 mL samples containing 1 M NaCl were dialyzed at room temperature using Slide-A-Lyzer Dialysis Flasks with 2K, 3.5K, 10K, or 20K MWCOs. The dialysis buffer (4 L) was changed after 2 and 5 hr (triangles) and at 41 hr for the flask with 2K MWCO. More than 95% of NaCl was removed within 8 to 18 hr (41 hr for the flask with 2K MWCO). A B C How-to video Watch the video to see how to use Slide-A-Lyzer G3 dialysis cassettes in a typical dialysis protocol. For more information, or to view additional products, go to thermofisher.com/dialysis Time (hr) Dialysis device in conical tube Conventional dialysis Residual NaCl (%) 0 25 50 75 100 Time (hr) Residual NaCl (M) 0 0 0.25 0.50 0.75 1.00 1.25 0 1 2 3 4 5 1 2 3 4 5 0 2 5 10 15 20 25 0 10 20 30 40 50 60 70 80 90 100 Time (hr) Residual NaCl (%) 2K 3.5K 10K 20K Protein sample preparation thermofisher.com/proteinbiology 19 Zeba desalting products Convenient spin formats help ensure rapid desalting with high protein recovery Thermo Scientific™ Zeba™ desalting products contain proprietary high-performance resins with exceptional desalting and protein recovery characteristics. They can help process even very dilute protein samples with high levels of protein recovery and greater than 95% retention (removal) of salts and other small molecules. The resin is provided in convenient spin columns, plates, and cartridges, for processing sample volumes between 2 μL and 4 mL (Tables 6–8). Highlights: • High performance—proprietary resin enables excellent protein recovery and efficient contaminant removal (Figure 6) • Flexible—available in spin columns, filter spin plates, and cartridges for a range of needs • Fast—no fraction screening or waiting for protein to emerge by gravity flow • Economical—cost-effective products that offer great performance Table 6. Zeba desalting products selection guide by format and recommended sample volume. Type Spin columns Spin plate Chromatography columns Format Micro 0.5 mL 2 mL 5 mL 10 mL 96-well 1 mL 5 mL Resin bed 75 μL 0.5 mL 2 mL 5 mL 10 mL 550 μL 1 mL 5 mL Sample volume (7K MWCO) 2–12 μL 30–130 μL 200–700 μL 500–2,000 μL 700–4,000 μL 20–100 μL 50–250 μL 100–1,500 μL Sample volume (40K MWCO) 5–14 μL 70–200 μL 200–900 μL 300–2,000 μL 1,000–4,000 μL 20–100 μL NA NA 20 Protein sample preparation thermofisher.com/proteinbiology Table 7. Thermo Scientific™ Zeba™ resin selection guide by protein recovery and small molecule removal. 7K MWCO 40K MWCO Size Recovery Removal Recovery Removal Peptide/protein <7 kDa NR* NR Protein 7–13 kDa ++ ++ Protein 14–20 kDa +++ +++ Protein 20–150 kDa +++ +++ Molecule <500 Da +++ +++ Molecule 600–1,200 Da ++ +++ Molecule 1,200–1,500 Da + ++ Molecule >1,500–2,000 Da NR + * NR: No recovery or removal. 1.5 mL 2.5 mL 3.5 mL 0.04 mg/mL BSA 0.2 mg/mL BSA 1.0 mg/mL BSA Thermo Fisher Scientific Load Cytiva spin Cytiva gravity Thermo Fisher Scientific Cytiva spin Cytiva gravity Thermo Fisher Scientific Cytiva spin Cytiva gravity Figure 6. Zeba Spin Desalting Columns result in high protein recovery while providing minimal sample dilution over a wider range of sample concentrations and volumes compared to alternative products. Zeba Spin Desalting Columns, 10 mL (7K MWCO), and Cytiva PD-10 Columns were used to desalt 1.5, 2.5, and 3.5 mL BSA samples at concentrations of 0.04, 0.2, and 1 mg/mL. Desalting was performed according to the manufacturers’ recommended protocols; both the spin and gravity protocols were used for the Cytiva PD-10 Columns. Protein recovery was analyzed by SDS-PAGE. For each electrophoresis gel, an aliquot of starting sample equal to 1 μg of BSA was loaded in lane 1 as the loading control; all other desalted samples were loaded in the gel at the same volume as the loading control. Differences in intensity between lanes are a combination of protein recovery and sample dilution caused by desalting. The largest differences in recovery and concentration were noticed in the outlined area. Table 8. Comparison of recommended sample volume capacities of common spin desalting products. Zeba spin desalting products Cytiva products Bio-Rad products 0 mL 0.01 mL 0.1 mL 0.5 mL 1 mL 2 mL 3 mL 4 mL Zeba micro spin column 0.5 mL Zeba spin column 2 mL Zeba spin column 5 mL Zeba spin column 10 mL Zeba spin column PD SpinTrap G-25 column PD MidiTrap G-25 column PD MiniTrap G-25 column PD-10 Desalting columns Micro Bio-Spin 6 column Bio-Spin 6 column For more information, or to view additional products, go to thermofisher.com/desalting Protein sample preparation thermofisher.com/proteinbiology 21 Protein concentrators Easy-to-use devices for rapid and efficient concentration Thermo Scientific™ Pierce™ Protein Concentrators are easy-to-use centrifugal devices that provide fast processing and excellent recovery of protein samples. These disposable ultrafiltration devices are available in four volume capacities (0.5, 6, 20, and 100 mL) and contain a polyethersulfone (PES) membrane in five distinct molecular weight cutoffs (MWCOs): 3K, 5K, 10K, 30K, 50K, and 100K, for the concentration, desalting, and buffer exchange of biological samples, such as tissue culture media and chromatography fractions, or to remove unincorporated label following protein bioconjugation reactions. Highlights: • Rapid processing—unique design minimizes membrane fouling • High recovery—retain >90% of protein samples while removing contaminants or exchanging buffers (Figure 7) • Convenient—clear markings, wide sample chamber, and removable filtrate chamber make handling simple and easy 1 mg/mL 0.05 mg/mL 1 mg/mL 0.05 mg/mL 1 mg/mL 0.05 mg/mL 0 20 40 30 10 60 80 100 90 70 50 Thermo Scientific Millipore Pall Protein recovery (%) 99 98 99 98 72 27 Figure 7. Pierce Protein Concentrators provide exceptional recovery of low-concentration samples. Samples of aprotinin (~6.5 kDa) were centrifuged in a Pierce Protein Concentrator, PES (3K MWCO, 0.5 mL), and other suppliers’ concentrators at 15,000 x g until the sample volume was 1/15 of the original volume. Protein recovery was determined using the Pierce BCA Protein Assay Kit. For more information, or to view additional products, go to thermofisher.com/concentrators 22 Protein sample preparation thermofisher.com/proteinbiology Protein quantitation products Protein assays Accurately quantifying total protein concentration is a key step in most experiments and workflows involving isolation, separation, and analysis of proteins by biochemical methods. Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration. Colorimetric and fluorescent reagent-based protein assay techniques are available. Protein is added to the reagent, producing a color change or fluorescent readout in proportion to the amount added. A standard curve consisting of known concentrations of a purified protein is used to determine protein concentration. Because of the diversity of protein samples, careful consideration should be given to the choice of assay. Important criteria for choosing an assay include: • Compatibility with the sample type and buffer components • Assay working range and required sample volume • Protein-to-protein uniformity • Speed and convenience for the number of samples to be tested • Availability of necessary equipment to measure the assay output Multiple formulations for flexible and accurate quantitation We offer numerous colorimetric and fluorescent assays for detection and quantitation of total protein to meet the sensitivity and compatibility requirements of your samples (Table 9). In addition, we offer easy-to-use colorimetric or fluorescent peptide assays that are designed specifically to improve the sensitivity and reproducibility of peptide mixture quantitation. For a full list of available protein concentration assays, visit thermofisher.com/proteinassays Table 9. Protein assay guide. Thermo Scientific™ Pierce™ Rapid Gold BCA Protein Assay Kit Thermo Scientific™ Pierce™ BCA Protein Assay Kit Thermo Scientific™ Pierce™ Coomassie Plus (Bradford) Assay Kit Invitrogen™ Qubit™ Protein BR Assay Kit Invitrogen™ NanoOrange™ Protein Quantitation Assay Assay highlights 5 min room temperature BCA assay Detergent-compatible assay with linear curve and high accuracy Reducing-agent compatible, rapid assay Rapid quantitation for broad range of protein concentrations Best for dilute samples or samples with limited volume Minimum sample volume 10 μL 25 μL 10 μL 10 μL 1 μL Working range 125–2,000 μg/mL 20–2,000 μg/mL 100–1,500 μg/mL 100–20,000 μg/mL 0.01–10 μg/mL Compatibility Detergents Detergents Reducing agents Detergents and reducing agents Reducing agents Incubation temperature RT 37°C RT RT 90–95°C Assay incubation time 5 min 30 min 5 min 10 min 10 min Assay wavelength 480 nm 562 nm 595 nm 470/570 nm 470/570 nm Required equipment Spectrophotometer or absorbance microplate reader Spectrophotometer or absorbance microplate reader Spectrophotometer or absorbance microplate reader Qubit 4 Fluorometer Fluorometer or fluorescence microplate reader Protein sample preparation thermofisher.com/proteinbiology 23 Figure 8. Accuracy of the Pierce Rapid Gold BCA Protein Assay and Bradford Protein Assay with known protein mixes. Both assays were conducted according to the respective manufacturers’ protocols, in a microplate format. For the Bradford assay, 10 μL of the BSA sample was added to 200 μL of the Bradford working reagent and incubated at room temperature for 5 minutes. For the Pierce Rapid Gold BCA Protein Assay, 20 μL of sample was added to 200 μL of Rapid Gold BCA working reagent and incubated at room temperature for 5 minutes. Known concentrations were based on manufacturers’ indicated concentrations and confirmed by absorbance at 280 nm. Figure 9. Standard curves for protein quantitation assays. Purified bovine serum albumin (BSA) in 0.9% saline (0–20 mg/mL) was used to generate standard curves for the Qubit Protein BR Assay, Pierce BCA Protein Assay, and the Bradford assay. Assays were conducted following the manufacturers’ protocols. The BCA and Bradford assays were performed in microplate format. Protein mix Concentration (mg/mL) Pierce Rapid Gold BCA Protein Assay Bradford Protein Assay (Bio-Rad) Known concentration A 0 0.1 0.3 0.4 0.6 0.9 0.8 1.0 0.5 0.7 0.2 B C D E F G H I 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 5,000 10,000 15,000 20,000 25,000 RFU Absorbance Sample concentration (μg/mL) Qubit Protein BR Assay Bradford BCA Broad dynamic range of the Qubit Protein BR Assay The Invitrogen™ Qubit™ Protein BR Assay is a fluorometric assay that combines accuracy, compatibility, and ease of use to detect total protein concentrations over a broad dynamic range. The broad linear response allows accurate determination of unknown protein concentrations and provides a higher dynamic range than other standard protein assays (Figure 9). The Qubit Protein BR Assay can be used to detect protein concentrations from 100 to 20,000 μg/mL and is compatible with many commonly used detergents and reducing agents. This allows most samples to be used neat (undiluted), eliminating the guesswork and dilution steps that accompany many protein quantitation methods. Accurate detergent-compatible Pierce BCA assays BCA protein assays have a unique advantage over Bradford (Coomassie dye–based) assays, as they are compatible with samples that contain up to 5% surfactants (detergents) and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy (Figure 8). 24 Protein sample preparation thermofisher.com/proteinbiology Qubit 4 Fluorometer The Qubit Protein BR Assay is optimized for use with the Invitrogen™ Qubit™ 4 Fluorometer. The Qubit 4 Fluorometer provides the combination of a user-friendly fluorometer and capability to run highly sensitive fluorescence-based quantitation assays. The Qubit 4 Fluorometer is a small, economical instrument designed to work seamlessly with Invitrogen™ Qubit™ assay kits for routine protein, DNA, and RNA quantitation, and RNA integrity and quality assessment. All settings and calculations are performed directly on the instrument and can be transferred by USB drive, USB cable, or by Wi-Fi. The system is simple, fast, and easy to use, yet enables consistently accurate results for subsequent applications. Only small sample volumes of 1–20 μL are required for all assays. Multiskan SkyHigh Microplate Spectrophotometer The Thermo Scientific™ Multiskan™ SkyHigh Microplate Spectrophotometer provides ease of use for all photometric protein quantitation measurements. The touchscreen models offer the flexibility to use the system as a stand-alone instrument, or in conjunction with Thermo Scientific™ SkanIt™ PC software. For direct 280 nm measurements, the system provides ready-made protocols for path length correction, which normalize the absorbance values of microvolumes to values measured in standard cuvettes. The colorimetric dye measurement is simple to perform with either internal user interface pre-built sessions or by downloading the pre-built session with all the data handling from the SkanIt Cloud Library. For more details, visit thermofisher.com/platereaders Test your protein research knowledge Question: Which of the following are important considerations when selecting an appropriate protein quantitation assay? A. Sample type and buffer component compatibility B. Assay working range and required sample volume C. Speed and convenience D. All of the above Answer: D Protein sample preparation thermofisher.com/proteinbiology 25 Ordering information Product Quantity Cat. No. Extraction Protein extraction reagents and subcellular fractionation kits M-PER Mammalian Protein Extraction Reagent 250 mL 78501 T-PER Tissue Protein Extraction Reagent 500 mL 78510 Pierce IP Lysis Buffer 100 mL 87787 RIPA Lysis and Extraction Buffer 250 mL 89901 Pierce IP Lysis Buffer 100 mL 87787 NE-PER Nuclear and Cytoplasmic Extraction Reagents 75 mL 78835 Mem-PER Plus Membrane Protein Extraction Kit 300 mL 89842 Mitochondria Isolation Kit for Cultured Cells 115 mL 89874 Subcellular Protein Fractionation Kit for Cultured Cells 35 mL 78840 Subcellular Protein Fractionation Kit for Tissues 115 mL 87790 Syn-PER Synaptic Protein Extraction Reagent 100 mL 87793 Pierce Cell Surface Protein Isolation Kit 8 reactions 89881 Lysosome Enrichment Kit for Tissues and Cultured Cells 230 mL 89839 Y-PER Yeast Protein Extraction Reagent 500 mL 78990 I-PER Insect Cell Protein Extraction Reagent 250 mL 89802 Pierce Plant Total Protein Extraction Kit 50 preparations A44056 B-PER Complete Bacterial Protein Extraction Reagent 250 mL 89821 Pierce Universal Nuclease for Cell Lysis 25 kU 88701 100 kU 88702 Pierce GPCR Extraction and Stabilization Reagent 100 mL A43436 N-PER Neuronal Protein Extraction Reagent 100 mL 87792 To view additional pack sizes and products, go to thermofisher.com/proteinextraction Detergents Tween 20 Surfact-Amps Detergent Solution 6 x 10 mL 28320 50 mL 85113 Tween 80 Surfact-Amps Detergent Solution 6 x 10 mL 28328 50 mL 28329 Triton X-100 Surfact-Amps Detergent Solution 6 x 10 mL 28314 50 mL 85111 Triton X-114 Surfact-Amps Detergent Solution 6 x 10 mL 28332 NP-40 Surfact-Amps Detergent Solution 6 x 10 mL 28324 50 mL 85124 Brij-35 Surfact-Amps Detergent Solution 6 x 10 mL 28316 50 mL 85117 Brij-58 Surfact-Amps Detergent Solution 6 x 10 mL 28336 To view additional pack sizes and products, go to thermofisher.com/detergents 26 Protein sample preparation thermofisher.com/proteinbiology Product Quantity Cat. No. Inhibitor cocktails and tablets Halt Protease Inhibitor Single-Use Cocktail (100X) 24 x 100 μL 78430 Halt Protease Inhibitor Cocktail (100X) 1 mL 87786 5 mL 78429 Halt Protease Inhibitor Single-Use Cocktail, EDTA-free (100X) 24 x 100 μL 78425 Halt Protease Inhibitor Cocktail, EDTA-free (100X) 1 mL 87785 5 mL 78437 Pierce Protease Inhibitor Mini Tablets 30 tablets A32953 Pierce Protease Inhibitor Tablets 20 tablets A32963 Pierce Protease Inhibitor Mini Tablets, EDTA-free 30 tablets A32955 Pierce Protease Inhibitor Tablets, EDTA-free 20 tablets A32965 Halt Phosphatase Inhibitor Single-Use Cocktail (100X) 24 x 100 μL 78428 Halt Phosphatase Inhibitor Cocktail (100X) 1 mL 78420 5 x 1 mL 78426 Pierce Phosphatase Inhibitor Mini Tablets 20 tablets A32957 Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100X) 24 x 100 μL 78442 Halt Protease and Phosphatase Inhibitor Cocktail (100X) 1 mL 78440 Halt Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-free (100X) 24 x 100 μL 78443 Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) 1 mL 78441 Pierce Protease and Phosphatase Inhibitor Mini Tablets 20 tablets A32959 Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free 20 tablets A32961 To view additional pack sizes and products, go to thermofisher.com/inhibitorcocktails Cleanup Dialysis devices, cassettes, and flasks Slide-A-Lyzer MINI Dialysis Device, 2K MWCO, 0.1 mL 50 devices 69580 Slide-A-Lyzer MINI Dialysis Device, 3.5K MWCO, 0.1 mL 50 devices 69550 Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 0.1 mL 50 devices 69570 Slide-A-Lyzer MINI Dialysis Device, 20K MWCO, 0.1 mL 50 devices 65950 Slide-A-Lyzer MINI Dialysis Device, 3.5K MWCO, 0.5 mL 25 devices 88400 Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 0.5 mL 25 devices 88401 Slide-A-Lyzer MINI Dialysis Device, 3.5K MWCO, 2 mL 25 devices 88403 Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 2 mL 25 devices 88404 Slide-A-Lyzer Dialysis Flask, 3.5K MWCO, 230 mL 4 flasks 87761 Slide-A-Lyzer Dialysis Flask, 10K MWCO, 250 mL 4 flasks 87762 Pierce 96-Well Microdialysis Plate, 0.1 mL, 10K MWCO 1 plate 88260 Pierce 96-Well Microdialysis Plate, 0.3 mL, 10K MWCO 1 plate A50469 Pierce 48-Well Microdialysis Plate, 1 mL, 10K MWCO 1 plate A50470 Slide-A-Lyzer G3 Dialysis Cassettes, 2K MWCO, 3 mL 10 cassettes A52961 Slide-A-Lyzer G3 Dialysis Cassettes, 3.5K MWCO, 3 mL 10 cassettes A52966 Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 3 mL 10 cassettes A52971 Slide-A-Lyzer G3 Dialysis Cassettes, 20K MWCO, 3 mL 10 cassettes A52976 Slide-A-Lyzer G3 Dialysis Cassettes, 3.5K MWCO, 15 mL 8 cassettes A52967 Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 15 mL 8 cassettes A52972 Slide-A-Lyzer G3 Dialysis Cassettes, 3.5K MWCO, 30 mL 6 cassettes A52968 Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 30 mL 6 cassettes A52973 Slide-A-Lyzer G3 Dialysis Cassettes, 3.5K MWCO, 70 mL 6 cassettes A52969 Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 70 mL 6 cassettes A52974 Slide-A-Lyzer G3 Dialysis Cassettes, 3.5K MWCO, 125 mL 6 cassettes A52970 Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 125 mL 6 cassettes A52975 To view additional pack sizes and MWCOs, go to thermofisher.com/dialysis Protein sample preparation thermofisher.com/proteinbiology 27 Product Quantity Cat. No. Desalting products Zeba Micro Spin Desalting Columns, 7K MWCO, 75 μL 25 columns 89877 Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL 25 columns 89882 Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL 25 columns 87766 Zeba Spin Desalting Columns, 7K MWCO, 2 mL 25 columns 89890 Zeba Spin Desalting Columns, 40K MWCO, 2 mL 25 columns 87769 Zeba Spin Desalting Columns, 7K MWCO, 5 mL 25 columns 89892 Zeba Spin Desalting Columns, 7K MWCO, 10 mL 25 columns 89894 Zeba 96-Well Spin Desalting Plates, 7K MWCO 2 plates 89807 Zeba Desalting Chromatography Cartridges, 7K MWCO, 1 mL 5 cartridges 89934 Zeba Desalting Chromatography Cartridges, 7K MWCO, 5 mL 5 cartridges 89935 Zeba Micro Spin Desalting Columns, 40K MWCO, 75 μL 25 columns 87764 To view additional pack sizes and MWCOs, go to thermofisher.com/desalting Protein concentrators Pierce Protein Concentrators PES, 3K MWCO, 0.5 mL 25 pack 88512 Pierce Protein Concentrators PES, 10K MWCO, 0.5 mL 25 pack 88513 Pierce Protein Concentrators PES, 30K MWCO, 0.5 mL 25 pack 88502 Pierce Protein Concentrators PES, 100K MWCO, 0.5 mL 25 pack 88503 Pierce Protein Concentrators PES, 3K MWCO, 2–6 mL 24 pack 88515 Pierce Protein Concentrators PES, 10K MWCO, 2–6 mL 24 pack 88517 Pierce Protein Concentrators PES, 30K MWCO, 2–6 mL 24 pack 88522 Pierce Protein Concentrators PES, 100K MWCO, 2–6 mL 24 pack 88524 Pierce Protein Concentrators PES, 10K MWCO, 5–20 mL 24 pack 88528 Pierce Protein Concentrators PES, 10K MWCO, 20–100 mL 24 pack 88535 Pierce Protein Concentrators PES, 50K MWCO, 5–20 mL 24 pack 88541 Pierce Protein Concentrators PES, 50K MWCO, 20–100 mL 4 pack 88542 To view additional pack sizes and MWCOs, go to thermofisher.com/concentrators Protein quantitation Protein assays Pierce Rapid Gold BCA Protein Assay Kit 250 mL A53226 500 mL A53225 Pierce BCA Protein Assay Kit 500 mL 23227 1 L 23225 Pierce Coomassie Plus (Bradford) Assay Kit 950 mL 23236 Qubit Protein BR Assay Kit 100 assays A50668 500 assays A50669 Qubit Protein BR Starter Kit 1 instrument and kit A51292 NanoOrange Protein Quantitation Kit 1 kit N6666 To view additional pack sizes and products, go to thermofisher.com/proteinassays 28 Protein sample preparation thermofisher.com/proteinbiology Protein purification Protein purification is essential to the study of protein function, structure, and interactions. A variety of methods can be used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. Common techniques include ion-exchange and affinity chromatography and immunoprecipitation. Protein purification products 30 IP, co-IP, and pull-down using magnetic beads 35 Magnetic stands 38 Ordering information 39 Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Protein purification thermofisher.com/proteinbiology 29 Protein purification and immunoprecipitation Ion exchange (IEX) and affinity chromatography are two strategies commonly used for partial or one-step protein purification. IEX chromatography enables the separation of proteins based on the protein charge at a particular pH. Since multiple proteins may have similar charges, IEX chromatography generally enables only partial purification of a protein of interest. Affinity chromatography is enabled by specific binding of a protein to an immobilized ligand. Because the protein of interest is tightly bound, contaminants can be removed through wash steps, and the bound protein can be stripped (eluted) from the support in a highly purified form. Affinity purification produces higher protein yields and requires fewer steps than other purification methods. Immunoprecipitation (IP) is small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support, and is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting or other techniques. Other similar techniques include co-immunoprecipitation (co-IP) and pull-down, which are similar to IP except that the target antigen is used to co-precipitate its binding partner(s) or associated protein complex from the lysate. Protein purification products High-performance resins and magnetic beads for maximum protein yield Our protein purification portfolio offers a broad range of products for ion exchange and affinity-based isolation of proteins and antibodies in microgram to kilogram quantities. Strong anion or cation exchange resins provide an intermediate level of purification during multi-step isolation or act as a polishing step during the final stages of purification. Biotinylated or recombinant proteins can be conveniently captured using avidin-based or affinity tag–based binding supports. Customized protein purification can be achieved by immobilizing ligands to the appropriate support. Accessory products are available for increased convenience, including disposable columns and binding and elution buffers. Rapid protein screening or IP, co-IP, and pull-down applications can be completed utilizing magnetic bead–based resins and kits, as described on pages 35–37. Highlights: • Broad product selection—strong ion exchange and affinity supports for purification and enrichment of proteins and antibodies; affinity ligands enable one-step purification of recombinant and biotinylated proteins, while activated supports provide a platform for custom protein immobilization (Table 1) • High performance—resins are designed to maximize protein yield and reduce background • More formats—magnetic beads, loose resins, FPLC cartridges, and 96-well filter plates enable protein purification from screening and small-scale phases to process-scale purification (Table 2) • Economical—pricing that is similar to or better than products from other leading suppliers 30 Protein purification thermofisher.com/proteinbiology Table 1. Overview of ion exchange, affinity, and activated supports. Application Purity level Ligand and/or chemistry Base bead type Packaging options Ion exchange purification Medium to high (application-specific) Strong anion exchange POROS Loose resin Strong cation exchange Antibody purification High Protein A, protein G, protein A/G Agarose, magnetic beads, magnetic agarose, POROS Loose resins or beads, spin columns and kits, chromatography cartridges, Protein L Agarose, magnetic beads 96-well spin plates Melon Gel Agarose Fusion protein purification High Ni-NTA, Ni-IDA, cobalt, glutathione Agarose, Superflow, magnetic beads, magnetic agarose Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates Anti–c-Myc, anti-HA, anti-FLAG Agarose, UltraLink magnetic beads Loose resins or beads, kits Biotin affinity purification High Avidin, streptavidin, NeutrAvidin, monomeric avidin Agarose, magnetic beads Loose resins, spin columns and kits, chromatography cartridges, 96-well spin plates Protein immobilization High Amine-reactive, sulfhydrylreactive, carbonyl-reactive, carboxyl-reactive Agarose Loose resins or dry powder Epoxy, tosyl-activated, carboxylic acid, amine Magnetic beads Loose beads Table 2. Select your resin based on purification scale and application. Scale High-throughput screening High-throughput batch Batch Pilot Process Description Small scale, automation compatible Lab or bench scale Lab or bench scale Scale-up desired Production scale Yield Microgram Milligram Milligram Gram Kilogram Format Magnetic particle processor Magnetic particle processor, 96-well spin plate (agarose) Gravity flow, spin column (agarose), fast protein liquid chromatography (FPLC) at low flow rates FPLC at medium flow rates FPLC at high flow rates Application High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis requiring mg scale Functional assays, structural analysis Structural analysis, intermediate-scale production Bulk production Recommended resin type Magnetic bead (1–2.8 μm) Magnetic agarose (10–40 μm) Agarose (45–165 μm) Superflow (45–165 μm) UltraLink resin (50–80 μm) POROS resin (50 μm) Protein purification thermofisher.com/proteinbiology 31 Table 3. Strong ion exchange purification selection guide. Chemistry Salt tolerance Recommended product High-throughput screening High-throughput batch Batch Pilot Process Strong anion exchange ≤25 mM Pierce Strong Anion Exchange Spin Columns • • ≤150 mM POROS HQ resin • • • ≤50 mM POROS XQ resin • • • Strong cation exchange ≤25 mM Pierce Strong Cation Exchange Spin Columns • • ≤150 mM POROS XS resin • • • Ion exchange chromatography resins and membranes We offer strong cation exchange (SCX) and strong anion exchange (SAX) resins composed of rigid polymeric beads with covalent surface chemistries. The robust manufacturing process yields outstanding physical and chemical stability, allowing easier handling and packing. These high-capacity chromatography media are designed to deliver excellent separation and scale-up capabilities. See Table 3 for recommended products based on application. Thermo Scientific™ Pierce™ Strong Cation or Anion Exchange Spin Columns are membrane-based centrifugal devices that eliminate the need for column packing, allow multiple samples to be processed simultaneously, and are ideal for working with low-volume buffer solutions. Affinity chromatography resins Our broad menu of resins and formats offers many options for single-step purification of biotinylated or recombinant proteins and antibodies. In addition, customized purification solutions can be designed by the covalent attachment of a ligand to one of our activated supports. Accessory products for all aspects of purification, including disposable columns and binding and elution buffers, are also available. 32 Protein purification thermofisher.com/proteinbiology Table 4. Antibody purification selection guide for Invitrogen™ and Thermo Scientific™ products. Mode Description Recommended product High-throughput screening High-throughput batch Batch Pilot Process Negative selection Removal of all nonimmunoglobulin proteins Melon Gel • IgG enrichment Immobilized immunoglobulin-binding proteins, to selectively remove IgG from a serum sample Dynabeads Protein A magnetic beads • Pierce High Capacity Protein A MagBeads, alkali stable • MabCaptureC High Capacity Protein A Resin, alkali stable • Pierce High Capacity Ni-IMAC MagBeads and Resin, EDTA compatible • Pierce Protein A Plus Agarose • POROS MabCapture A Select Resin • • • Dynabeads Protein G magnetic beads • Pierce Protein G Plus Agarose • POROS MabCapture G Select Resin • • • Pierce Protein A/G Magnetic Beads • Pierce Protein A/G Magnetic Agarose • Pierce Protein A/G Plus Agarose • POROS MabCapture A/G Select Resin • • • Pierce Protein L Magnetic Beads • Pierce Protein L Agarose • IgG enrichment Thiophilic adsorption Pierce Thiophilic Adsorbent • IgM enrichment Immobilized mannanbinding protein (MBP) Pierce Mannan Binding Protein Agarose • IgA enrichment Immobilized jacalin, a D-galactose–binding lectin Pierce Jacalin Agarose • Antibody purification Proteins A, G, A/G, and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). These and other ligands enable purification of general immunoglobulins from a crude sample (Table 4). Depending on the sample source, an antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2–5% of total IgG in mouse serum is specific for the antigen used to immunize the animal. Thermo Scientific™ POROS™ MabCapture™ Select resins demonstrate outstanding binding capacity at a range of flow rates, making them suitable for large-scale bioprocessing needs (Figure 1). Figure 1. Comparison of dynamic binding capacity vs. flow rate. Each column (0.5 cm ID x 5 cm) was packed with 1 mL of resin and was challenged with human IgG (1 mg/mL) at flow rates of 500, 300, or 150 cm/hr (corresponding to residence times of 0.3, 1, and 2 min, respectively). The dynamic binding capacity (total protein loaded) was determined at 10% breakthrough. (A) Comparison between Thermo Scientific™ POROS™ MabCapture™ G Select and Cytiva™ Protein G Sepharose™ 4 Fast Flow resins. (B) Comparison between Thermo Scientific™ POROS™ MabCapture™ A/G Select and Thermo Scientific™ Pierce™ Protein A/G Plus resins. Linear flow rate (cm/hr) IgG bound per mL of resin (mg) 0 5 10 15 20 500 300 150 POROS MabCapture A/G Select resin Pierce Protein A/G Plus agarose POROS MabCapture G Select resin Protein G Sepharose 4 Fast Flow resin Linear flow rate (cm/hr) IgG bound per mL of resin (mg) 0 15 10 5 20 25 30 500 300 150 Linear flow rate (cm/hr) IgG bound per mL of resin (mg) 0 5 10 15 20 500 300 150 POROS MabCapture A/G Select resin Pierce Protein A/G Plus agarose POROS MabCapture G Select resin Protein G Sepharose 4 Fast Flow resin Linear flow rate (cm/hr) IgG bound per mL of resin (mg) 0 15 10 5 20 25 30 500 300 150 A B Protein purification thermofisher.com/proteinbiology 33 Recombinant protein purification We offer a variety of resins for the purification of recombinant proteins. These resins are available in multiple formats to accommodate a variety of needs, from high-throughput screening to batch and pilot-scale purification. Superflow resins have undergone extensive chemical characterization. We have ligands targeting a variety of fusion tags, including 6xHis, GST, FLAG™ epitope, c-Myc, and HA (Table 5). Table 5. Thermo Scientific™ recombinant protein purification selection guide. Tag Ligand Features Recommended product High-throughput screening High-throughput batch Batch Pilot DYKDDDDK (FLAG) Anti-FLAG Immobilized antibody Pierce Anti-DYKDDDDK Magnetic Agarose • Pierce Anti-DYKDDDDK Affinity Resin (UltraLink resin) • • c-Myc Anti–c-Myc Immobilized antibody Pierce Anti–c-Myc Magnetic Beads • Pierce Anti–c-Myc Agarose (Superflow) • • HA Anti-HA Immobilized antibody Pierce Anti-HA Magnetic Beads • Pierce Anti-HA Agarose • 6xHis Ni-NTA or Ni-IDA Higher protein yield Pierce Ni-NTA Magnetic Agarose Beads • ProBond Nickel Chelating Resin • HisPur Ni-NTA Agarose Resin • HisPur Ni-NTA Superflow Resin • Cobalt Higher protein purity Dynabeads His-Tag Isolation Magnetic Beads • HisPur Cobalt Agarose Resin • HisPur Cobalt Superflow Resin • GST Glutathione Solubility and purification tag Pierce Glutathione Magnetic Agarose Beads • Pierce Glutathione Agarose • Pierce Glutathione Superflow Agarose • For more information, or to view additional products, go to thermofisher.com/proteinpurification 34 Protein purification thermofisher.com/proteinbiology Immunoprecipitation (IP), co-IP, and pull-down using magnetic beads Fast, reproducible sample processing with high yield and low nonspecific binding Magnetic beads are the most rapidly growing method for IP and pull-down assays because they are faster, easier, and more efficient for pulling down proteins than nonmagnetic methods. We offer a wide variety of conjugated magnetic beads, including the highly referenced Invitrogen™ Dynabeads™ magnetic beads and the economical Thermo Scientific™ Pierce™ magnetic beads or magnetic agarose, to meet most application and budget needs. Highlights: • Low background—little to no nonspecific binding, and no preclearing • Highly sensitive—magnetic beads are the ideal choice for sensitive applications, such as IP of low-abundance proteins • Antibody savings—all binding occurs on the smooth outer surface of the beads, conserving precious antibodies and providing a solution that is cost-efficient per sample • Fast and easy—magnetic beads offer a rapid IP protocol, with no centrifugation or preclearing steps (Figure 2) • Flexible—products for IP, co-IP, and pull-down assays; ideal for both manual and automated protocols • Compatible—magnetic beads can be used in multiple workflows, including western blotting, mass spectrometry, and qPCR (for ChIP analysis) • Automation ready—Dynabeads products for IP can be optimized with a Thermo Scientific™ KingFisher™ purification system to decrease hands-on preparation time and increase sample output (Figures 3 and 4) Figure 2. Immunoprecipitation in <40 minutes. Dynabeads magnetic beads precoupled with protein A or protein G act as a suspendable solid support that can be fixed by the use of a magnet. This allows for simple and efficient antibody capture, followed by immunoprecipitation of pure target peptides, proteins, protein complexes, or other antigens. Start with: Figure 3. Thermo Scientific™ KingFisher™ Apex instrument. For more information about the KingFisher automation system, or to schedule a demo, go to thermofisher.com/kingfisherapex KingFisher Apex KingFisher Flex Manual 16 kDa 36 50 26 Std I II III I II III I II III CD81 Figure 4. IP of CD81 using manual methods vs. Thermo Scientific™ KingFisher™ Flex and Apex instruments. Jurkat cells expressing CD81 were lysed and incubated with Dynabeads Protein G coated with anti-CD81 antibody using manual and automated methods in triplicate. The samples were prepared for electrophoresis and western blotting, and labeling was performed using the anti-CD81 antibody. Protein purification thermofisher.com/proteinbiology 35 The optimal product for IP, co-IP, and pull-down assays depends on the antibody or recombinant protein in the application (Table 6). Table 6. Choose your isolation strategy and find your product. Choose this if you use: Surface coating or ligand on the magnetic beads Target Nonspecific binding IP protocol time Main benefits for IP Thermo Scientific™ and Invitrogen™ products Unconjugated primary antibody Protein A, G, A/G, or L Primary antibodies from most species; proteins A, G, and L bind different antibody species and subclasses with different specificities Low Dynabeads magnetic beads: <40 min Pierce beads: 130–180 min • Dynabeads magnetic beads— fastest, easiest protocol with low nonspecific binding and high yield and reproducibility • Pierce Magnetic IP-MS Kit (Protein A/G) validated for mass spectrometry workflows • Pierce Crosslink kit includes DSS crosslinker Dynabeads Protein A Dynabeads Protein A Immunoprecipitation Kit Dynabeads Protein G Dynabeads Protein G Immunoprecipitation Kit Pierce Classic Magnetic IP/Co-IP Kit Pierce Protein A/G Magnetic Beads Pierce Crosslink Magnetic IP/Co-IP Kit Pierce Magnetic IP-MS Kit (Protein A/G) Pierce Protein L Magnetic Beads Secondary antibodies Mouse IgG or rabbit IgG Low Dynabeads magnetic beads: <40 min • Fast and easy protocol • Low nonspecific binding • Specific binding of mouse or rabbit IgGs Dynabeads M-280 Sheep Anti–Mouse IgG Dynabeads M-280 Sheep Anti–Rabbit IgG Epoxy- and NHSactivated beads Any protein ligand (e.g., antibody, peptide) Ultralow Dynabeads magnetic beads: • Ab coupling time: overnight • Co-IP protocol time: 30–40 min Pierce beads: • Ab coupling time: 30– 60 min • Protocol time: 120 min • Covalent coupling of the Ab gives ultralow nonspecific binding • No need for crosslinking • Gentle and efficient co-IP of even large protein complexes Dynabeads Antibody Coupling Kit Dynabeads Co-Immunoprecipitation Kit Pierce NHS-activated magnetic beads Biotinylated antibody Streptavidin Any biotinylated antibody or ligand Low Dynabeads magnetic beads: <40 min Pierce beads: 60–120 min • Binds any biotinylated protein • For samples high in soluble IgGs • Recombinant Ab lacking the Fc region Dynabeads MyOne Streptavidin T1 Dynabeads M-280 Streptavidin Pierce Streptavidin Magnetic Beads Pierce Magnetic IP-MS Kit (Streptavidin) Recombinant protein Fusion tags Different beads bind proteins with the following tags: His, GST, DYKDDDDK (FLAG), HA, c-Myc Low Dynabeads His-tag beads: ~25 min Pierce beads: ~70 min • Purify many different proteins incorporated with the same tag • No need for antibodies Dynabeads His-Tag Isolation and Pulldown Pierce Anti-DYKDDDDK Magnetic Agarose Pierce HA-Tag Magnetic IP/Co-IP Kit Pierce c-Myc-Tag Magnetic IP/Co-IP Kit Choose these products if you use unconjugated primary antibodies—your choice of antibody-binding products depends on your downstream application, or if you do not want the antibody co-eluted with your target protein. Protein A, G, and A/G beads are most commonly used for IP and co-IP applications since unconjugated primary antibodies that face towards the protein target bind directly to the coated beads in a short and simple incubation step. Epoxy beads are often used to obtain ultralow nonspecific binding, or to avoid crosslinking, since the antibody is covalently coupled to the beads and not eluted with the target protein. The Invitrogen™ epoxy beads and co-IP kit (including optimized buffers) are recommended for co-IP applications involving larger protein complexes. Choose these products if you use biotinylated antibodies—your best choice when using a biotinylated antibody with streptavidin-coated beads for IP. These products are also useful in the following cases: • If you have a sample rich in soluble IgGs • If you have a recombinant antibody lacking Fc regions • If you want to use streptavidin magnetic beads for pull-down applications using a biotinylated protein as bait Choose these products if you have a recombinant protein with a fusion tag—the most popular fusion tags for recombinant protein expression are covered by Pierce and Dynabeads products. DYKDDDDK (FLAG epitope), HA-tag, and c-Myc tags are ideal for IP/co-IP applications, while His-tag and GST-tagged proteins are for pull-down assays. 36 Protein purification thermofisher.com/proteinbiology Figures 5–8 show example data from experiments using various isolation strategies. Figure 6. Higher IP yield with Protein A/G beads. U2OS (human osteosarcoma) cells were lysed in IP Lysis/Wash Buffer, and incubated with and without anti-Cdk1 antibody overnight at 4°C. Thermo Scientific™ Pierce™ Protein A/G Magnetic Beads were compared to Mag Sepharose™ Beads (Cytiva) and PureProteome™ Protein A and Protein G products (MilliporeSigma). The beads were washed multiple times using a KingFisher Flex instrument and then eluted with SDS-PAGE reducing sample buffer for 10 minutes at room temperature. The eluates were resolved by SDS-PAGE and analyzed by western blot for Cdk1. Figure 7. The Thermo Scientific™ Pierce™ MS-Compatible Magnetic IP Kit (streptavidin) allows for effective target capture and elution. Percentages indicate elution efficiency compared to bead boil. The eluates were analyzed by western blot. Antibodies were labeled with the Thermo Scientific™ Pierce™ Antibody Biotinylation Kit for IP and used with the Pierce MS-Compatible Magnetic IP Kit to immunoprecipitate target proteins from cell lysates. 25 kDa Dynabeads Protein G 10 min Dynabeads Protein G 60 min Cytiva Protein G 10 min Cytiva Protein G 60 min Bio-Rad Qiagen MilliporeSigma Figure 5. Protein yields determined by western blot analysis. Invitrogen™ Dynabeads™ Protein G magnetic beads have the best overall performance for yield, capacity, and nonspecific binding. Thermo Scientific Pierce Cytiva A/G G A MilliporeSigma G A U2OS cell lysate 47.7 – 31.5 – 24.4 – – Cdk1 (~34 kDa) Load MS elution Bead boil Load MS elution Bead boil PP2A (72%) PTEN (90%) EGFR (80%) Stat1 (88%) -catenin (140%) HDAC1 (140%) P13K (247%) pan Akt (97%) Figure 8. Comparison of protein purification using Thermo Scientific™ Pierce™ Anti-DYKDDDDK Affinity Resin and another supplier’s product. C-terminal DYKDDDDK-tagged GFP protein was expressed using the Thermo Scientific™ 1-Step Human High-Yield Maxi In Vitro Translation (IVT) Kit and immunoprecipitated using Pierce Anti-DYKDDDDK Affinity Resin or Anti-FLAG™ M2 Affinity Gel (MilliporeSigma). Tagged proteins were competitively eluted with Pierce 3x DYKDDDDK Peptide and analyzed by western blot. Comparison of the starting lysate (L), flow-through (FT), eluate (E), and bead-boiled sample (BB) shows effective capture and elution of DYKDDDDK-tagged proteins. L FT E BB FT E BB Thermo Scientific FT E BB MilliporeSigma To learn more about other magnetic bead products to help isolate everything from cells to exosomes, download the Dynabeads magnetic beads brochure at thermofisher.com/dynabeads Protein purification thermofisher.com/proteinbiology 37 Magnetic stands Multiple formats for low- to high-throughput sample processing Invitrogen™ DynaMag™ magnets isolate any target in combination with Dynabeads magnetic beads. Reduce waiting time as these powerful magnets quickly pull the bead-bound target to the tube wall. The DynaMag magnets help to ensure optimal working positions and are functionally adapted to suit various workflows. Highlights: • Optimized—developed and certified for use with Dynabeads magnetic beads • Easy to handle—designed with ergonomics in mind • More choices—different formats to accommodate different volume and throughput needs The Invitrogen™ DynaMag™-2 Magnet is our most commonly used magnet for immunoprecipitation and holds up to 16 standard 1.5–2 mL tubes in numbered spaces. The optimal working volume is 10–2,000 μL. There are eight positions on each side of the rack. The top rack can be quickly removed from the magnet in the base, ready for vortexing, rotation, or manual sample shaking. A center pin in the rack ensures equal vortexing of all tubes, providing excellent control and visibility during sample processing. Invitrogen™ DynaMag™-Spin Magnet, which accommodates up to six (1.5 mL) microcentrifuge tubes, supports a working volume of 10–1,500 μL. The circular top rack can be quickly removed from the magnet in the base, ready for vortexing or manual sample shaking. Plate-based magnetic stands, such as the Invitrogen™ DynaMag™-96 series, are ideal for manual and automated work, with a footprint the same size as that of a 96-well plate. The recommended working volume is 5–200 μL. DynaMag-2 Magnet DynaMag-Spin Magnet Sample rack for DynaMag-2 Magnet DynaMag-96 Side Magnet DynaMag-96 Bottom Magnet DynaMag-96 Side Skirted Magnet Test your protein research knowledge Question: For large yields of highly purified protein, which purification strategy is the best? A. Immunoprecipitation B. Ion exchange chromatography C. Affinity chromatography D. Co-immunoprecipitation Answer: C 38 Protein purification thermofisher.com/proteinbiology Ordering information Product Quantity Cat. No. Purification Strong cation exchange purification resins POROS XS Resin 10 mL 82071 Strong anion exchange purification resins POROS XQ Resin 10 mL 82073 POROS HQ Resin 10 mL 82077 Antibody purification resins Pierce Protein A Plus Agarose 5 mL 22811 MabCaptureC High Capacity Protein A Resin 1 mL A53031 Pierce High-Capacity Protein A MagBeads, alkali stable 1 mL A53035 POROS MabCapture A Select Resin 15 mL 82080 Pierce Protein G Plus Agarose 2 mL 22851 POROS MabCapture G Select Resin 15 mL 82083 Pierce Protein A/G Magnetic Agarose Beads 1 mL 78609 Pierce Protein A/G Plus Agarose 2 mL 20423 POROS MabCapture A/G Select Resin 15 mL 82086 Pierce Protein L Agarose 2 mL 20510 Melon Gel Monoclonal IgG Purification Kit 1 kit 45214 Recombinant protein purification resins and magnetic beads Pierce High-Capacity Ni-IMAC Resin, EDTA compatible 1 mL A50588 HisPur Ni-NTA Magnetic Beads 2 mL 88831 Pierce Ni-NTA Magnetic Agarose Beads 1 mL 78605 HisPur Ni-NTA Agarose Resin 10 mL 88221 HisPur Ni-NTA Superflow Agarose 10 mL 25214 HisPur Cobalt Agarose Resin 10 mL 89964 HisPur Cobalt Superflow Agarose 10 mL 25228 Pierce Glutathione Magnetic Agarose Beads 1 mL 78601 Pierce Glutathione Agarose 10 mL 16100 Pierce Glutathione Superflow Agarose 10 mL 25236 Pierce Anti-DYKDDDDK Affinity Resin 1 mL settled A36801 Pierce Anti-c-Myc Agarose 2 mL 20168 Pierce Anti-HA Agarose 1 mL 26181 Biotin binding purification resins and magnetic beads Pierce Streptavidin Magnetic Beads 1 mL 88816 Pierce High Capacity Streptavidin Agarose 2 mL 20357 Pierce High Capacity NeutrAvidin Agarose 5 mL 29202 Pierce Monomeric Avidin Agarose 5 mL 20228 Activated support resins and magnetic beads Pierce NHS-Activated Agarose, Dry 1 g 26196 AminoLink Plus Coupling Resin 10 mL 20501 SulfoLink Coupling Resin 10 mL 20401 CarboxyLink Coupling Resin 25 mL 20266 GlycoLink Immobilization Kit 10 columns 88941 Pierce NHS-Activated Magnetic Beads 1 mL 88826 Dynabeads M-270 Epoxy 60 mg 14301 Dynabeads MyOne Tosylactivated 2 mL 65501 Dynabeads M-270 Amine 2 mL 14307D To view additional pack sizes and products, go to thermofisher.com/proteinpurification Protein purification thermofisher.com/proteinbiology 39 Product Quantity Cat. No. Immunoprecipitation Immunoprecipitation using magnetic beads Dynabeads Protein A 1 mL 10001D Dynabeads Protein A Immunoprecipitation Kit 2 mL 10006D Dynabeads Protein A IP Kit and Magnet Starter Pack 40 reactions 10018D Dynabeads Protein A and Magnet Starter Pack 40 reactions 10013D Dynabeads Protein G 1 mL 10003D Dynabeads Protein G Immunoprecipitation Kit 40 reactions 10007D Dynabeads Protein G IP Kit and Magnet Starter Pack 40 reactions 10019D Dynabeads Protein G and Magnet Starter Pack 40 reactions 10014D Dynabeads Protein A/Protein G and Magnet Starter Pack 40 reactions 10015D Pierce Protein A/G Magnetic Beads 1 mL 88802 Pierce Classic Magnetic IP/Co-IP Kit 40 reactions 88804 Pierce Crosslink Magnetic IP/Co-IP Kit 40 reactions 88805 Pierce MS-Compatible Magnetic IP Kit (Protein A/G) 40 reactions 90409 Pierce Protein L Magnetic Beads 1 mL 88849 Dynabeads M-280 Sheep Anti-Mouse IgG 2 mL 11201D Dynabeads M-280 Sheep Anti-Rabbit IgG 2 mL 11203D Dynabeads Antibody Coupling Kit 1 kit 14311D Dynabeads Co-Immunoprecipitation Kit 40 reactions 14321D Pierce Direct Magnetic IP/Co-IP Kit 40 reactions 88828 Dynabeads M-280 Streptavidin 2 mL 60210 Dynabeads MyOne Streptavidin C1 2 mL 65001 Pierce MS-Compatible Magnetic IP Kit (Streptavidin) 40 reactions 90408 Dynabeads His-Tag Isolation and Pulldown 2 mL 10103D Pierce Anti-DYKDDDDK Magnetic Agarose 1 mL A36797 Pierce HA-Tag Magnetic IP/Co-IP Kit 40 reactions 88838 Pierce c-Myc-Tag Magnetic IP/Co-IP Kit 40 reactions 88844 Immunoprecipitation kits using agarose resin Pierce Classic IP Kit 50 reactions 26146 Pierce Crosslink IP Kit 50 reactions 26147 Pierce Direct IP Kit 50 reactions 26148 Pierce Co-Immunoprecipitation Kit 50 reactions 26149 GlycoLink IP Kit 25 reactions 88943 Pierce Biotinylated Protein Interaction Pull-Down Kit 25 reactions 21115 EZ-Link Desthiobiotinylation and Pull-Down Kit 5 reactions 16138 Pierce c-Myc-Tag IP/Co-IP Kit 25 reactions 23620 Pierce HA-Tag IP/Co-IP Kit 25 reactions 26180 Pierce GST Protein Interaction Pull-Down Kit 25 reactions 21516 Pierce His Protein Interaction Pull-Down Kit 25 reactions 21277 To view additional pack sizes and products, go to thermofisher.com/immunoprecipitation 40 Protein purification thermofisher.com/proteinbiology Protein gel electrophoresis Gel electrophoresis is a technique in which charged molecules are separated according to physical properties such as charge or mass as they are forced through a sieving gel matrix by an electrical current. Proteins are commonly separated in this manner using polyacrylamide gel electrophoresis (PAGE) to identify individual proteins in complex samples or to examine multiple proteins within a single sample. SureCast Gel Handcast Station 45 Bolt Bis-Tris Plus and NuPAGE Bis-Tris mini gels 45 Novex Tris-glycine gels 47 Electrophoresis chambers 50 PowerEase Touch Power Supplies 51 Protein ladders and standards 52 Protein stains 54 Ordering information 55 Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Protein gel electrophoresis thermofisher.com/proteinbiology 41 Several forms of PAGE exist, each providing different types of information about the proteins under investigation. Nondenaturing PAGE, also called native PAGE, separates proteins based on their charge and size, preserving their conformation and activity. Denaturing and reducing SDS-PAGE, the most widely used electrophoresis technique, separates proteins by mass. Two-dimensional (2D) PAGE separates proteins by isoelectric point in the first dimension and by mass in the second dimension. SDS-PAGE separates proteins primarily by mass. The ionic detergent sodium dodecyl sulfate (SDS) binds to the proteins in the mixture shielding their respective charge and making them all uniformly negatively charged. Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode. Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel matrix. Once separated by electrophoresis, proteins can be detected in a gel with various stains, transferred onto a membrane for detection by western blotting, or excised and extracted for analysis by mass spectrometry. Protein gel electrophoresis is, therefore, a common step in many kinds of proteomic analyses. Gel matrix Acrylamide is the base component used in electrophoretic gels to separate proteins by size; when mixed with bisacrylamide in the presence of ammonium persulfate, it forms a crosslinked polymer network. Ammonium persulfate acts as a polymerizing agent by producing free radicals, while TEMED (N,N,N´,N´-tetramethyl ethylenediamine) is added to catalyze and speed up the reaction. The size of the pores created in the gel is inversely related to the amount of acrylamide and the bisacrylamide to acrylamide ratio. Electrophoresis gels are formulated in buffers that conduct an electrical current through the matrix. The solution is poured into the thin space between two glass or plastic plates of an assembly called a cassette. Once the gel polymerizes, the cassette is mounted into an apparatus so that opposite edges (top and bottom) are placed in contact with buffer chambers containing a cathode and anode, respectively. When proteins are added in wells at the top edge and current is applied, the proteins are drawn by the current through the matrix slab and separated by its sieving properties. To obtain optimal resolution of proteins, a stacking gel is cast over the top of the resolving gel. The stacking gel has a lower concentration of acrylamide, lower pH, and a different ionic content. This allows the proteins in a loaded sample to be concentrated into a tight band during the first few minutes of electrophoresis before entering the resolving portion of a gel. A stacking gel is not necessary when using a gradient gel, as the gradient itself performs this function. Linear vs. gradient gels Gels that have a single acrylamide percentage are referred to as linear gels, and those with a range are referred to as gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins than does a linear gel. Mini vs. midi protein gels Commercial gels are available in two size formats: mini gels and midi gels. Both gels have similar run lengths, but midi gels are wider than mini gels, allowing midi gels to have more wells or larger wells. The additional wells in the midi gels permit more samples or large sample volumes to be loaded onto one gel. Homemade vs. precast gels Traditionally, researchers poured their own gels using standard recipes that are widely available in protein method books. Most laboratories now depend on the convenience and consistency afforded by commercially available ready-to-use precast gels. Precast gels are available in a variety of percentages; these include difficult-to-pour gradient gels that provide excellent resolution and separate proteins over the widest possible range of molecular weights. Technological innovations in buffer and gel polymerization methods enable manufacturers to produce gels with greater uniformity and longer shelf life than with traditional equipment and methods. In addition, precast polyacrylamide gels obviate the need to work with the acrylamide monomer—a known neurotoxin and suspected carcinogen. Protein standards To assess the relative molecular weight (MW) of a protein on a gel, protein MW markers are run in the outer lanes of the gel. A standard curve can be constructed using the distances migrated by each marker protein. The distance migrated by the unknown protein is then plotted, and the molecular weight is interpolated from the standard curve. Several kinds of ready-to-use protein MW markers are available that are labeled or prestained for different modes of detection. These are pre-reduced and, therefore, primarily suited for SDS-PAGE rather than native PAGE. MW markers are detectable via their specialized labels or by ordinary protein staining methods. 42 Protein gel electrophoresis thermofisher.com/proteinbiology Overview of precast gels Invitrogen™ precast gels offer convenience, speed, and consistency. We offer precast gels in four different formulations and a wide variety of percentages, gradients, and sample well configurations. The choice of whether to use one formulation or another depends on the abundance of the protein you are separating, the size of the protein, and your downstream application. Four gel options to fit your protein separation needs: • Bis-Tris gels for broad-range, low-abundance protein separation, or for downstream applications requiring high protein integrity, such as posttranslational modification analysis, mass spectrometry, or sequencing • Tris-glycine gels for broad-range, high-abundance protein separation • Tris-acetate gels for high molecular weight protein separation, up to 500 kDa • Tricine gels for low molecular weight protein and peptide separation Find out more at thermofisher.com/proteingels Protein gel performance guarantee We stand behind the quality of our high-performance protein gels. Purchase Invitrogen™ protein gels with confidence, knowing that our gels are backed by our protein gel performance guarantee. If an Invitrogen protein gel does not perform in your experiment as described on our website or Certificate of Analysis, we will replace the product at no cost to you, or we will provide you with a credit for future purchase. Learn more at thermofisher.com/proteingelguarantee Protein gel welcome packs Invitrogen™ protein gel welcome packs contain the components for outstanding protein separation and are available for mini- and midi-format protein gels. The typical protein gel welcome pack provides all of the necessary gels, buffers, and reagents you need, as well as an Invitrogen™ Mini Gel Tank, Invitrogen™ XCell SureLock™ Mini-Cell, or Invitrogen™ SureLock™ Tandem Midi Gel Tank. Protein gel welcome packs are also available in mini gel and midi gel formats designed for use in protein expression experiments and subsequent purification steps. Find out more at thermofisher.com/proteingelwelcome Protein gel electrophoresis thermofisher.com/proteinbiology 43 Isoelectric point Molecular weight Protease activity Novex WedgeWell Tris-glycine gels with native buers NativePAGE gels 1st dimension 2nd dimension ZOOM IPG strips Novex Zymogram gels (casein, blue casein, or gelatin substrates) Novex IEF gels Novex WedgeWell Tris-glycine gels, 2D well NuPAGE Bis-Tris gels, 2D well Novex WedgeWell Tris-glycine ZOOM gels, IPG well Novex WedgeWell Tris-glycine gels, 2D well NuPAGE Bis-Tris ZOOM gels, IPG well NuPAGE Bis-Tris gels, 2D well SureCast Gel Handcast System* SureCast Gel Handcast System* SureCast Gel Handcast System* SureCast Gel Handcast System* Molecular weight Low molecular weight proteins and peptides (2.5–40 kDa) Novex tricine gels NuPAGE Bis-Tris gels NuPAGE Bis-Tris gels Bolt Bis-Tris Plus gels NuPAGE Bis-Tris gels Bolt Bis-Tris Plus gels Bolt Bis-Tris Plus gels Bolt Bis-Tris Plus gels Novex WedgeWell Tris-glycine gels SureCast Gel Handcast System* NuPAGE Tris-acetate gels Application NuPAGE Bis-Tris and Novex Tris-glycine midi gels Downstream applications requiring high protein integrity (e.g., posttranslational modifications, mass spectrometry) Large sample volume for high-detection sensitivity High-abundance protein, Coomassie dye, or silver staining High-sensitivity western blotting Low throughput Medium or high throughput High molecular weight proteins (40–500 kDa) Broad-range molecular weight separation (6–400 kDa) Find the right gel using our interactive gel selection tool at thermofisher.com/proteingelguide Denaturing separation Native separation * Pour your own Tris-glycine gels with this system. Gel selection guide Find the right gel for your research needs based on molecular weight, downstream applications, and throughput requirements. 44 Protein gel electrophoresis thermofisher.com/proteinbiology SureCast Gel Handcast Station Pour your own gels The Invitrogen™ SureCast™ Gel Handcast Station is designed for 100% leak-free protein gel casting. The SureCast system is fully compatible with our Mini Gel Tank. Benefits of the SureCast Gel Handcast System include: • Leak-free design—gels that are more usable; less wasted time • Superior glass plate durability—up to 20 times more durable than other suppliers’ plates • Unique tilt feature—helps minimize spillage when pouring acrylamide solutions • Simple assembly of casting components—uses a singlemotion, load-and-lock mechanism Use Invitrogen™ SureCast™ handcasting reagents as well as other popular polyacrylamide gel casting reagents. Neutral-pH gel systems for optimal separation Invitrogen™ Bolt™ Bis-Tris Plus and NuPAGE™ Bis-Tris gels are precast polyacrylamide gels designed for optimal separation of a broad molecular weight range of proteins under denaturing conditions during gel electrophoresis. These gels help deliver consistent performance with a neutral-pH environment to minimize protein degradation, resulting in sharper bands without typical gel “smiling”. Additionally, preserving protein integrity becomes particularly important when separating low-abundance proteins. The unique wedge-well design of Bolt Bis-Tris Plus gels allows loading up to twice the sample volume of other precast gels. In addition, Bolt gels can be run in as little as 20 minutes. Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels are ideal for western blot transfer and analysis along with any other technique where protein integrity is crucial. Bolt Bis-Tris Plus gels are available in the mini-gel format and NuPAGE Bis-Tris gels are available in mini- and midi-gel formats, as well as multiple thicknesses. Bolt Bis-Tris Plus and NuPAGE Bis-Tris mini gels Protein gel electrophoresis thermofisher.com/proteinbiology 45 Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels offer: • Preserved protein integrity—neutral-pH formulation minimizes protein modifications and degradation (Figure 1) • Superior band quality and band volume—Bis-Tris gels are designed to deliver sharp, straight bands with higher band volume (Figure 2) • High sample volume capacity—wedge-well design of Bolt Bis-Tris Plus mini gels allows detection of proteins in very dilute samples or measurement of low-abundance proteins • More efficient western blot transfer—neutral pH prevents reoxidation of reduced samples during protein transfer • High lot-to-lot consistency—coefficient of variation (CV) of only 2% for Rf values (migration) • Long shelf life—16 months at room temperature EPHB3 GST HCK FLT1 IKKβ MAPK14 DDR2 MagicMark standard Figure 1. Bolt Bis-Tris Plus mini gels help provide better western blotting results. A western blot of a Bolt gel shows clean, sharp protein signals corresponding to only full-length proteins, whereas a western blot of a Bio-Rad™ TGX™ gel shows multiple low molecular weight degradation products. Protein kinases implicated in cancer (IKKβ, EPHB3, HCK, MAPK14, FLT1, and DDR2) were analyzed on a Bolt Bis-Tris Plus gel and a Bio-Rad TGX Tris-glycine gel. The purified kinases (50 ng each), along with Invitrogen™ MagicMark™ XP Western Protein Standard and purified recombinant GST protein, were loaded on a 10-well, 4–12% Bolt gel and a 10-well, 4–20% Bio-Rad TGX gel. The samples were separated and transferred to 0.45 μm PVDF membranes using the respective manufacturers’ protocols. Immunodetection was performed using an anti-GST antibody and Invitrogen™ WesternBreeze™ chemiluminescence detection. The blots were imaged using an LAS-1000 system (FujiFilm). MagicMark standard EPHB3 GST HCK FLT1 MAPK14 DDR2 IKKβ Bolt Bis-Tris Plus gel Bio-Rad TGX Tris-glycine gel Find out more at thermofisher.com/nupage Figure 2. Publication-quality protein electrophoresis gel results using a NuPAGE Bis-Tris 4–12% gradient midi gel and the SureLock Tandem Midi Gel Tank. Lanes 1, 20: 5 μL Thermo Scientific™ PageRuler™ Broad Range Unstained Protein Ladder; lanes 2–7: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, 1 μg of HeLa lysate; lanes 8, 13: 5 μL Invitrogen™ Novex™ Mark12™ Unstained Standard; lanes 9–12: 240 ng, 180 ng, 120 ng, 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14–19: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, 1 μg of E. coli lysate. The gel was run in Invitrogen™ NuPAGE™ MOPS running buffer, and stained with Invitrogen™ SimplyBlue™ SafeStain. NuPAGE Bis-Tris 4–12% gradient midi gel 46 Protein gel electrophoresis thermofisher.com/proteinbiology Novex Tris-glycine gels Load up to 60 μL of sample The Invitrogen™ Novex™ Tris-Glycine mini gels, WedgeWell™ format, and Invitrogen™ Novex™ Tris-Glycine Plus midi gels are polyacrylamide gels based on traditional Laemmli chemistry that enable the use of Laemmli sample and running buffers. Novex Tris-Glycine gels provide high-quality performance and separation of a wide range of proteins into well-resolved bands. Highlights: • Wedge-shaped wells—easily load up to 60 μL of sample without sacrificing gel width or length (mini gel only) • High performance—excellent protein band resolution and sharpness (Figure 3) • Fast run conditions—quickly separate your proteins using constant voltage in less than 60 minutes • Flexible—Novex Tris-glycine gels do not contain SDS and can be used to run proteins in native or in denatured form Novex 4–20% Tris-glycine mini gel, WedgeWell format Bio-Rad TGX 4–20% gel 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Novex 4–20% Tris-glycine mini gel, WedgeWell format Bio-Rad TGX 4–20% gel 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Figure 3. Better protein resolution and band sharpness with Novex Tris-glycine mini gels, WedgeWell format. Protein ladder, purified proteins, and E. coli lysate were loaded on a gradient Novex 4−20% Tris-Glycine mini gel, WedgeWell format, and a Bio-Rad TGX 4–20% gradient gel. The Bio-Rad TGX gel displays numerous low molecular weight protein degradation products below major bands in lanes 3, 4, 7, and 8 that are not seen in the Novex Tris-glycine gel. The Novex gel also displays better protein band sharpness and resolution of lysate than the Bio-Rad gel. Lanes 1, 10: 5 μL Invitrogen™ Mark12™ Unstained Standard; lane 2: 10 μg E. coli lysate (10 μL); lane 3: 6 μg catalase (10 μL); lane 4: 6 μg carbonic anhydrase (10 μL); lane 5: 6 μg lysozyme (10 μL); lane 6: 6 μg hIgM (10 μL); lane 7: 6 μg BSA (10 μL); lane 8: 6 μg β-galactosidase (10 μL); lane 9: 20 μg E. coli lysate (20 μL). Find out more at thermofisher.com/novexwedge Protein gel electrophoresis thermofisher.com/proteinbiology 47 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 260 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 160 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 6 kDa 6 kDa 6 kDa 6 kDa 6 kDa 2.5 kDa 2.5 kDa 3.5 kDa 2.5 kDa 3.5 kDa 3.5 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 80 kDa 260 kDa 160 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 10 20 30 40 50 60 70 80 90 %oflengthofgel 0 8% MES 8% MOPS 10% MES 10% MOPS 12% MES 12% MOPS 4–12% MES 4–12% MOPS 8% MES 8% MOPS 10% MES 10% MOPS 4–12% MES 4–12% MOPS 12% MES Denaturingseparation Denaturingseparation Invitrogen™ Bolt™ Bis-Tris Plus Gels Invitrogen™ NuPAGE™ Bis-Tris Gels Protein mini gel migration chart Use this chart to compare migration patterns of proteins of various molecular weights across the Invitrogen™ precast gel product line. 48 Protein gel electrophoresis thermofisher.com/proteinbiology 97 kDa 97 kDa 200 kDa 116 kDa 66 kDa 66 kDa 55 kDa 97 kDa 116 kDa 66 kDa 55 kDa 55 kDa 36 kDa 36 kDa 31 kDa 6 kDa 240 kDa 290 kDa 500 kDa 160 kDa 40 kDa 97 kDa 116 kDa 66 kDa 55 kDa 240 kDa 290 kDa 500 kDa 160 kDa 40 kDa 14 kDa 14 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 200 kDa 116 kDa 31 kDa 21 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 14 kDa 97 kDa 66 kDa 66 kDa 55 kDa 36 kDa 200 kDa 1,048 kDa 1,048 kDa 720 kDa 720 kDa 480 kDa 480 kDa 242 kDa 242 kDa 146 kDa 1,048 kDa 1,236 kDa 720 kDa 480 kDa 242 kDa 146 kDa 66 kDa 1,048 kDa 720 kDa 480 kDa 242 kDa 146 kDa 116 kDa 31 kDa 21 kDa 6 kDa 14 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 20 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 97 kDa 200 kDa 116 kDa 66 kDa 55 kDa 36 kDa 31 kDa 14 kDa 6 kDa 2.5 kDa 3.5 kDa 21 kDa 97 kDa 200 kDa 116 kDa 66 kDa 55 kDa 36 kDa 31 kDa 14 kDa 6 kDa 2.5 kDa 3.5 kDa 21 kDa 12% MOPS 10% 12% 4–12% 8–16% 4–20% 3–8% 7% 10% 16% 10–20% 3–8% 7% 3–12% 4–16% Denaturingseparation Denaturing separation Native separation Native separation Blotting and sequencing Synthetic peptides and tryptic analysis Wide range NuPAGE Tris-Acetate Gels Invitrogen™ NativePAGE™ Gels Invitrogen™ Novex™ Tris-Glycine Gels, WedgeWell™ format Invitrogen™ Novex™ Tricine Gels Invitrogen™ NuPAGE™ Tris-Acetate Gels Protein gel electrophoresis thermofisher.com/proteinbiology 49 Electrophoresis chambers Ideal for multiple applications and needs The Invitrogen™ gel portfolio offers four gel electrophoresis chamber systems to help you achieve your application and throughput needs (Table 1). The Invitrogen™ Mini Gel Tank is designed for more intuitive use and greater convenience compared to traditional electrophoresis tanks. The unique side-by-side tank design allows you to perform electrophoresis of 1 or 2 mini gels, and less running buffer is required because the gel chambers are separated. The Invitrogen™ XCell4 SureLock™ Midi-Cell Electrophoresis System is ideal for higher throughput experiments. The user-friendly design allows electrophoresis of up to 4 midi gels simultaneously. Highlights: • Flexible—run 1–4 gels simultaneously, depending on the chamber • Optimized—chambers amenable to wet transfer or midi-gel applications • Unique—Mini Gel Tank enables side-by-side gel loading and enhanced viewing during use Watch our Mini Gel Tank video at thermofisher.com/minigeltank Find out more at thermofisher.com/electrophoresischambers Table 1. Electrophoresis chamber systems. Mini Gel Tank XCell SureLock Mini-Cell SureLock Tandem Midi Gel Tank XCell4 SureLock Midi-Cell Gel capacity Up to 2 mini gels Up to 2 mini gels Up to 2 midi gels Up to 4 midi gels Cell dimensions (L x W x H; height with lid on) 32 x 11.5 x 16 cm 14 x 13 x 16 cm 25 x 17.9 x 17.3 cm 21 x 19 x 16 cm Advantages • The Mini Gel Tank is versatile and compatible with all Invitrogen precast and handcast mini gels; the unique tank design enables convenient side-by-side gel loading and enhanced viewing during use • Mini Blot Module is available for wet protein transfers • Instrument incorporates a gel tension wedge in place of the rear wedge used on earlier models • XCell II Blot Module is available for wet protein transfers • Easy-to-use apparatus, with separate chambers for each gel, enabling scalable buffer usage • SureLock Tandem Midi Blot Module is available for wet protein transfers • Advanced apparatus for easier, more reliable electrophoresis with midi gels 50 Protein gel electrophoresis thermofisher.com/proteinbiology PowerEase Touch Power Supplies Easy touchscreen programming and operation The Invitrogen™ PowerEase™ Touch Power Supplies make setting up custom protocols or selecting one of the several preprogrammed gel electrophoresis and transfer methods a breeze with an improved 4.3-inch backlit LCD touchscreen display and user interface. The power supplies are ideal for DNA or RNA electrophoresis, SDS-PAGE, native PAGE, isoelectric focusing (IEF), and 2D electrophoresis. The PowerEase Touch Power Supplies offer four sets of output jacks that can be used simultaneously and three modes: constant voltage, constant current, and constant power for flexibility of use and efficiency. The sturdy polyurethane feet and stackable housing design allow stacking of power supplies for a reduced footprint on the lab bench. • Ease of use—LCD touchscreen display and user interface show clear menu prompts for easy use by hand or stylus and convenient monitoring of run progress • Convenient—four sets of output terminals allow running of multiple electrophoresis units • Customizable—program up to 100 custom methods, 20 steps per method, 999 minutes per step; or select one of several preprogrammed Invitrogen gel electrophoresis and transfer methods • Safety—features automatic detection of No Load, Over Temperature, Over Voltage, Over Current, Load Change, and Ground Leak The PowerEase Touch 120W Power Supply can be a cost-saving solution for mini gel separation and transfer with a maximum output of 300 V and 500 mA. The PowerEase Touch 350W Power Supply is a medium-throughput power supply supporting mini and midi gel runs and transfers, with a maximum output of 300 V and 3 A (3,000 mA). The PowerEase Touch 600W Power Supply is our highest-throughput model, allowing up to 14 midi gel runs and 4 midi gel transfers with an output of up to 3 A and 500 V. The PowerEase Touch HV Power Supply is a high-voltage power supply developed to support the entire 2D electrophoresis workflow, featuring a voltage output of 3,500 V and a current output of up to 500 mA. Protein gel electrophoresis thermofisher.com/proteinbiology 51 Protein ladders and standards Prestained and unstained formats with exceptional lot-to-lot consistency We offer a broad range of prestained and unstained protein ladders in a ready-to-use format to facilitate easy protein analysis during gel electrophoresis and western blotting (Tables 2 and 3). Highlights: • Performance—sharp protein bands and consistent migration patterns provide easy molecular weight determination (Figure 4) • Convenience—protein ladders are ready to load, with no heating required • Reliability—exceptional lot-to-lot consistency and reproducibility kDa 260 140 100 70 50 40 35 25 10 15 20 30 40 50 60 80 100 220 120 kDa 70 50 40 kDa 300 250 180 130 100 kDa 250 130 95 72 55 36 28 17 10 PageRuler Plus Prestained Protein Ladder Spectra Multicolor High Range Protein Ladder iBright Prestained Protein Ladder Spectra Multicolor Broad Range Protein Ladder MagicMark XP Western Protein Standard Table 2. Prestained and unstained protein ladders. Prestained protein ladders Low range Spectra Multicolor Low Range Protein Ladder Broad range PageRuler Plus Prestained Protein Ladder High range HiMark Prestained Protein Standard Recommended for: • Determining approximate molecular weight • Monitoring the progress of electrophoresis runs • Estimating the efficiency of protein transfer to the membrane during western blotting Unstained protein ladders Low range PageRuler Unstained Low Range Protein Ladder Broad range PageRuler Unstained Broad Range Protein Ladder High range HiMark Unstained Protein Standard Recommended for: • Determining precise molecular weight of target protein Other Western blot MagicMark XP Western Protein Standard Specialty iBright Prestained Protein Ladder PageRuler Prestained NIR Protein Ladder BenchMark Fluorescent Protein Standard BenchMark His-tagged Protein Standard IEF Marker 3–10 Find out more at thermofisher.com/proteinladders 250 130 95 70 55 80 30 43 34 26 15 10 kDa kDa Figure 4. Migration patterns of protein ladders. Thermo Scientific™ PageRuler™ and Spectra™ ladders are visualized directly after gel separation. The Invitrogen™ MagicMark™ XP standard is visualized by chemiluminescent substrate detection. The Invitrogen™ iBright™ Prestained Protein Ladder has three methods of detection. Left: prestained ladder visualized after gel separation; center: chemiluminescent substrate detection of western blot; right: near-IR fluorescence detection of western blot. 52 Protein gel electrophoresis thermofisher.com/proteinbiology Table 3. Selection guide for protein ladders and standards. Category Product Advantages Range No. of bands Recommended for Unstained ladders and standard Unstained standards PageRuler Unstained Low Range Protein Ladder Sharp bands and precise molecular weight estimation for low molecular weight proteins 3.4–100 kDa 8 MW determination, gel staining, chemiluminescent band visualization PageRuler Unstained Broad Range Protein Ladder Sharp bands and precise molecular weight estimation for a wide range of proteins 5–250 kDa 14 MW determination, gel staining, chemiluminescent band visualization HiMark Unstained Protein Standard Convenient molecular weight estimation for high molecular weight proteins 20–1,200 kDa 8 MW determination (native gels), gel staining, chemiluminescent band visualization Prestained protein ladders and standard Prestained standards iBright Prestained Protein Ladder • Fluorescence visualization—detect the ten stained proteins using the 670 nm red laser or 700 nm channel of a fluorescence imager • Western blot confirmation—detect the two unstained proteins using the detection system for the target protein • Reference band—55 kDa band has greater intensity for easy orientation 11–250 kDa 12 Western blotting: detection of the two unstained bands using the detection method for the target protein; compatible with chemiluminescent substrates and fluorescent secondary antibodies PageRuler Prestained Protein Ladder Outstanding clarity for easy molecular weight determination of low molecular weight proteins 10–180 kDa 10 MW determination, protein band visualization, monitoring electrophoresis run and transfer, chemiluminescent band visualization PageRuler Plus Prestained Protein Ladder Outstanding clarity for easy molecular weight determination of a broad range of proteins 10–250 kDa 9 MW determination, protein band visualization, monitoring electrophoresis run and protein transfer HiMark Prestained Protein Standard Superb analysis of high molecular weight proteins 30–460 kDa 9 MW determination (high MW proteins), protein band visualization, monitoring electrophoresis run and protein transfer Spectra Multicolor Broad Range Protein Ladder Superior visualization and analysis of a broad range of proteins 10–260 kDa 10 MW determination, protein band visualization (best), monitoring electrophoresis run and protein transfer Spectra Multicolor High Range Protein Ladder Superior and convenient visualization of high molecular weight proteins 40–300 kDa 8 MW determination, protein band visualization (best), monitoring electrophoresis run and protein transfer Other ladders and standards Chemiluminescent standard MagicMark XP Western Protein Standard Accurate molecular weight estimation directly on western blots 20–220 kDa 9 MW determination, gel staining, chemiluminescent band visualization (best) Near-infrared (NIR) standard PageRuler Prestained NIR Protein Ladder Sharp prestained standard for near-IR fluorescent visualization and protein sizing 11–250 kDa 10 MW determination Fluorescent standard BenchMark Fluorescent Protein Standard Efficient estimation of molecular weight by fluorescent detection 11–155 kDa 7 MW determination, chemiluminescent band visualization His-tag standard BenchMark His-tagged Protein Standard Convenient detection and protein sizing of His-tagged proteins 10–160 kDa 10 MW determination, gel staining, chemiluminescent band visualization (using anti-6xHis antibody) IEF IEF Marker 3–10 Accurate determination of protein isoelectric points pI 3.5–10.7 13 pI determination Protein gel electrophoresis thermofisher.com/proteinbiology 53 Protein stains Optimized formulations for specific applications Once protein bands have been separated by electrophoresis, they can be directly visualized using different methods of in-gel detection. Over the past several decades, demands for improved sensitivity for small sample sizes and compatibility with downstream applications and detection instrumentation have driven the development of several basic staining methods. Each method has particular advantages and disadvantages, and a number of specific formulations of each type of method provide optimal performance for various situations. Our portfolio includes Coomassie, silver, fluorescent, and specialty gel stains (Table 4). Highlights: • Convenient—most formulations are ready to use • Optimized—reagents and kits developed for specific applications and workflows • Flexible offering—multiple options to meet sensitivity or budget needs Table 4. Gel stain selection guide. Protein staining Coomassie staining Silver staining Fluorescent protein staining Sensitivity 25 ng 0.5 ng 0.5 ng Ease of use +++ + + Mode of action In acidic buffer conditions, Coomassie stain binds to basic and hydrophobic residues of proteins, changing from dull reddish-brown to intense blue. Silver ions interact and bind with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). Silver ions are reduced to metallic silver, resulting in a brown-black color. Most fluorescent stains involve simple dye-binding mechanisms rather than chemical reactions that alter protein functional groups. Detection Visual Visual Compatible imaging system Compatibility with downstream applications Compatible with mass spectrometry (MS) and sequencing Certain formulations are compatible with MS Most stains are compatible with MS Products Value: PageBlue Protein Staining Solution Performance: SimplyBlue SafeStain Premium: Imperial Protein Stain Value: Pierce Silver Stain Kit Performance: SilverXpress Silver Staining Kit Mass spectrometry: Pierce Silver Stain for Mass Spectrometry Value: SYPRO Red Protein Gel Stain Performance: SYPRO Orange Protein Gel Stain Premium: SYPRO Ruby Protein Gel Stain Test your protein research knowledge Question: How does the composition of a stacking gel differ from that of a resolving gel? A. A stacking gel has a higher concentration of acrylamide than a resolving gel. B. A stacking gel has a lower pH than a resolving gel. C. There is no difference in composition between the two gels. D. A stacking gel has the same ionic content as a resolving gel. Answer: B 54 Protein gel electrophoresis thermofisher.com/proteinbiology Ordering information Product Quantity Cat. No. SureCast welcome packs SureCast Gel Handcast Bundle A—Hardware and Reagents 1 kit HC1000SR SureCast Gel Handcast Bundle B—Hardware Only 1 kit HC1000S SureCast Gel Handcast Station SureCast Gel Handcast Station 1 casting station HC1000 To view additional products, go to thermofisher.com/surecast Bolt Bis-Tris Plus gels Bolt Bis-Tris Plus mini gels* Multiple Multiple Bolt welcome pack Multiple Multiple Each welcome pack includes: • Mini Gel Tank • 2 boxes of Bolt Gels (2 boxes, 20 gels) • Bolt MES Running Buffer (20X), Bolt LDS Sample Buffer (4X) • Bolt Sample Reducing Agent (10X) • PageRuler Plus Prestained Ladder, 10 to 250 kDa * One box contains 10 gels. To view additional products, go to thermofisher.com/bolt NuPAGE Bis-Tris gels NuPAGE Bis-Tris mini gels Multiple Multiple NuPAGE Bis-Tris midi gels Multiple Multiple NuPAGE Tris-acetate mini gels Multiple Multiple NuPAGE Tris-acetate midi gels Multiple Multiple NuPAGE Bis-Tris Welcome Pack Multiple Multiple To view products, go to thermofisher.com/nupage Novex Tris-glycine gels Novex Tris-glycine gels, WedgeWell format Multiple Multiple Novex Tris-glycine plus midi gels Multiple Multiple Novex Tris-glycine midi or mini welcome packs Multiple Multiple Each welcome pack includes: • Novex Tris-Glycine Gels, WedgeWell format, or Novex Tris-Glycine Plus Midi Gels, 2 boxes • Novex Tris-Glycine SDS Running Buffer (10X), 500 mL • Novex Tris-Glycine SDS Sample Buffer (2X), 20 mL • NuPAGE Sample Reducing Agent (10X), 250 μL • PageRuler Plus Prestained Protein Ladder, 10–250 kDa, 2 x 250 μL • Mini Gel Tank or SureLock Tandem Midi Gel Tank To view additional products, go to thermofisher.com/novexwedge To view all protein gels welcome packs, visit thermofisher.com/proteingelwelcome Protein standards PageRuler Plus Prestained Protein Ladder 2 x 250 μL 26619 PageRuler Prestained Protein Ladder 2 x 250 μL 26616 Spectra Multicolor Broad Range Protein Ladder 2 x 250 μL 26634 Spectra Multicolor High Range Protein Ladder 2 x 250 μL 26625 HiMark Prestained Protein Standard 250 μL LC5699 PageRuler Unstained Protein Ladder 2 x 250 μL 26614 PageRuler Unstained Low Range Protein Ladder 2 x 250 μL 26632 iBright Prestained Protein Ladder 2 x 250 μL LC5615 MagicMark XP Western Protein Standard 250 μL LC5602 NativeMark Unstained Protein Standard 5 x 50 μL LC0725 To view additional products, go to thermofisher.com/proteinladders Protein gel electrophoresis thermofisher.com/proteinbiology 55 Product Quantity Cat. No. Gel stains PageBlue Protein Staining Solution 1 L 24620 SimplyBlue SafeStain 1 L LC6060 Imperial Protein Stain 1 L 24615 Pierce Silver Stain Kit 1 L 24612 SilverXpress Silver Staining Kit 1 kit LC6100 SYPRO Orange Protein Gel Stain 500 μL S6650 SYPRO Red Protein Gel Stain 500 μL S6653 SYPRO Ruby Protein Gel Stain 1 L S12000 Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kit 1 kit P21875 Pro-Q Diamond Phosphoprotein Gel Stain 1 L P33300 To view additional products, go to thermofisher.com/proteinstains Electrophoresis chamber systems and power supplies Mini Gel Tank 1 unit A25977 XCell SureLock Mini-Cell 1 unit EI0001 SureLock Tandem Midi Gel Tank 1 each STM1001 XCell4 SureLock Midi-Cell 1 each WR0100 PowerEase Touch 120W Power Supply (115 VAC) 1 each PS0120 PowerEase Touch 350W Power Supply (115 VAC) 1 each PS0350 PowerEase Touch 600W Power Supply (115 VAC) 1 each PS0600 PowerEase Touch HV Power Supply (115 VAC) 1 each PSHV00 To view additional products, go to thermofisher.com/chambersystems To view all electrophoresis power supplies, go to thermofisher.com/powersupplies 56 Protein gel electrophoresis thermofisher.com/proteinbiology Western blotting Western blotting was introduced in the late 1970s and is now a routine and fundamental technique for protein analysis. Western blotting, also called protein blotting or immunoblotting, uses antibodies to identify specific protein targets after they have been separated by electrophoresis and transferred to a membrane. The specificity of the antibody–antigen interaction enables a target protein to be detected in a complex protein mixture, such as cell or tissue lysate. Western blotting can be used to generate qualitative and semi-quantitative data for a protein of interest. Wet transfer systems 60 Rapid transfer systems 62 Western blot detection 64 Ordering information 72 Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Western blotting thermofisher.com/proteinbiology 57 Transfer Following electrophoresis, the proteins must be transferred from the polyacrylamide gel to a membrane, typically using electroelution, or electrophoretic transfer, because of its speed and transfer efficiency. This method uses the electrophoretic mobility of proteins to transfer them from the gel to the membrane. Electrophoretic transfer of proteins involves placing the gel in direct contact with a piece of nitrocellulose or other suitable protein-binding support and sandwiching this between two electrodes submerged in a conducting solution. When an electric field is applied, the proteins move out of the gel and onto the surface of the membrane, where the proteins become tightly attached. The result is a membrane with a copy of the protein pattern that was originally in the gel. Efficiency can vary dramatically among proteins; it also depends on factors such as the composition of the gel, degree of contact of the gel with the membrane, the position of the electrodes, the transfer time, size and composition of proteins, field strength, and the presence of detergents and alcohols in the buffer. Blocking and washing The membranes used in western blotting have a high affinity for proteins. Therefore, after the transfer of proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of detection antibodies during subsequent steps. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio. Use of antibodies Western blotting is typically performed by probing the blocked membrane with a primary antibody that recognizes a specific protein or epitope. The choice of primary antibody for a western blot will depend on the antigen to be detected and the availability of antibodies for that antigen that have been verified for western blotting. In general, a primary antibody that recognizes the target protein is not directly detectable. Therefore, tagged secondary antibodies or other detection reagents are used as the means of ultimately detecting the target protein (indirect detection). A wide variety of labeled secondary detection reagents can be used; the choice of which depends upon either the species of animal in which the primary antibody was raised (the host species) or any tag on that antibody (e.g., biotin). Following the blocking and antibody incubations steps, extensive washing is necessary to remove unbound reagents and reduce background, thereby increasing the signal-to-noise ratio. Insufficient washing will result in high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot. As with other steps in western blotting, a variety of buffers may be used. Detection step While there are many different tags that can be conjugated to a secondary or primary antibody, the detection method used will limit the choice of what can be used in a western blotting assay. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic, and chemiluminescent substrates are available for use with either enzyme. HRP-conjugated antibodies, which generally use chemiluminescent substrates, are considered superior to AP-conjugated antibodies with respect to the specific activities of both the enzyme and antibody, due to the smaller size of HRP and compatibility with conjugation reactions. In addition, the high activity rate, good stability, low cost, and wide availability of substrates make HRP the enzyme of choice for most applications. In well-optimized assays, chemiluminescent reactions can produce stable light output for 1–24 hours, depending on the substrate, allowing consistent and sensitive detection that may be documented with X-ray film or digital imaging equipment. The use of fluorophore-conjugated antibodies requires fewer steps because there is no substrate development step in the assay. Recent advances in digital imaging and development of new fluorophores have increased the sensitivity and popularity of using fluorescent probes for western blotting and other immunoassays. This method has the added advantage of multiplex compatibility (using more than one fluorophore in the same experiment). 58 Western blotting thermofisher.com/proteinbiology Overview of western blot transfer systems When choosing a transfer methodology, convenience, speed, flexibility, reproducibility, and throughput are important considerations (Table 1). While the devices the laboratory currently has might be convenient, consider how alternative methods may improve steps such as transfer preparation and cleanup. A rapid dry transfer system, which utilizes a unique gel matrix that incorporates buffer ions instead of transfer buffer–soaked filter papers, with ready-to-use consumables, can provide convenience, requiring very little setup and cleanup time. Regarding transfer speed, wet transfer systems are typically the slowest, followed by conventional semi-dry transfer (using Towbin buffer), with dry transfer systems being the fastest. However, with the specially designed transfer buffers for semi-dry systems, semi-dry systems can match the speeds offered by dry transfer systems. Wet tank systems come in a variety of capacities and Table 1. Characteristics of Invitrogen™ transfer systems. Wet transfer Semi-dry transfer Dry transfer Mini Blot Module SureLock Tandem Midi Blot Module Power Blotter Systems iBlot 3 Western Blot Transfer Device Capacity 1 mini gel per blot module; 1–2 blot modules per tank 1 midi gel per blot module; 1–2 blot modules per tank 1–4 mini or 1–2 midi gels 1–4 mini or 1–2 midi gels Transfer time 60 min 30 min 7–10 min 3–8 min Blotting area 9 x 9 cm 9.2 x 14.4 cm 10 x 18 cm or 21 x 22.5 cm Station 1: 8.5 x 17 cm Station 2: 8.5 x 17 cm Transfer buffer volume 220 mL per blot module 300 mL per blot module Pre-cut membranes and filters: 50–100 mL; pre-assembled Select transfer stacks: buffer not required Buffer not required Power supply External External Internal Internal Built-in cooling – – – Yes Required equipment Mini Gel Tank SureLock Tandem Midi Gel Tank – – Learn more about the western blot transfer process in the Western Blotting Handbook at thermofisher.com/westernhandbook designs and typically offer the greatest flexibility in throughput, but this attribute should be weighed against the transfer speed and potential handling inconsistencies. With respect to transfer efficiency, these modern transfer systems will transfer proteins of typical molecular weights with high efficiency. Our semidry systems and dry system offer preprogrammed optimized methods for particular molecular weight ranges, including high and low molecular weights. Transferring very high or very low molecular weight proteins often requires optimization regardless of the system used. However, because of the flexibility of wet transfer (e.g., transfer membranes of different pore sizes can be swapped easily and transfer buffer formulations can be modified), wet tank transfer is a good place to start when transferring proteins of very high or low molecular weights. Western blotting thermofisher.com/proteinbiology 59 Wet transfer systems Mini Blot Module The Invitrogen™ Mini Blot Module is a wet transfer device used exclusively with the Mini Gel Tank and is designed to make your western blot transfers simple and easy to perform. The tank accommodates one Mini Blot Module per chamber, or two blot modules total in the side-by-side layout. The universal connection and molded gasket make the blot module easy to use, while the inner core of the blot module allows for use of less methanol-based transfer buffer per wet transfer than other commercially available transfer systems. At the recommended conditions and constant voltage, proteins can typically be transferred to nitrocellulose or PVDF membranes in 30 to 60 minutes. Highlights: • Universal module design—allows modules to fit in either chamber of the tank, simplifying the transfer setup • Unique gasket seal—helps prevent buffer leakage to minimize mess during setup of your western blot transfer • 1/2-inch buffer chamber—requires only 220 mL per blot of methanol-based transfer buffer, helping save you money on buffer and disposal costs • Standard 60-minute transfer protocol—accelerates your workflow so you can get results faster • Robust electrodes, sturdy steel plates—for highly efficient and reliable western blot transfers View the how-to video: 60 Western blotting thermofisher.com/proteinbiology SureLock Tandem Midi Blot Module The Invitrogen™ SureLock™ Tandem Midi Gel Tank is uniquely designed to enable convenient, reliable gel electrophoresis and protein transfer using high-performance Invitrogen™ midi gels. With the SureLock Tandem Midi Blot Module, the tank performs efficient room temperature wet transfers for downstream western blot analysis in 30 minutes. The tank accommodates two blot modules, allowing transfer of one or two gels at a time. The unique design of the SureLock Tandem Midi Blot Module uses considerably less transfer buffer (only ~300 mL per transfer) than other midi-size wet transfer systems. This lower buffer requirement reduces the amount of hazardous methanol waste. Highlights: • 2-in-1 midi gel electrophoresis and transfer tank—separate and transfer proteins in the same tank using high-performance Invitrogen midi gels • Two separate gel chambers—run 1 or 2 gels or transfers using only the necessary amount of buffer for each gel, minimizing buffer cost and waste • Optimal performance with fast transfer protocols—efficient, room temperature transfers in 30 minutes; eliminate the need to prechill buffers and the hassle and messiness of ice baths View the how-to video: Learn more at thermofisher.com/wettransfer Western blotting thermofisher.com/proteinbiology 61 Rapid transfer systems iBlot 3 western blot transfer system The next generation of protein transfer performance and convenience The Invitrogen™ iBlot™ 3 Western Blot Transfer system is a next-generation dry protein transfer device that delivers high performance and convenience along with ready-touse consumables. Perform reliable, high-quality transfers fast—in as little as three minutes. In addition, two independently controlled transfer stations allow you to share the device with your coworker, simultaneously running a total of four mini gels or two midi gels with two different programs. The preassembled single-use consumables and ultrafast transfer methods eliminate variability—helping ensure consistent and repeatable results. In addition, the robust design features built-in cooling that reduces heat buildup and facilitates run-to-run consistency. Better transfer efficiency With the iBlot 3 device, you can achieve equivalent or better protein transfer efficiency compared with wet-tank transfer and other rapid transfer methods (Figure 1). The design offers unique built-in adjustable cooling so that you can optimize the transfer of both high- and low-molecular weight proteins. Learn more at thermofisher.com/iblot3 Figure 1. The iBlot 3 system demonstrates superior transfer efficiency. (A) Hsp70: Invitrogen™ NuPAGE™ 4 to 12% Bis-Tris mini gels were loaded with 20, 15, 10, 5, 2.5, 1.25, 0.625, and 0.312 μg of A431 lysate per lane, from left to right. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Immunoprocessing was completed using the Invitrogen™ Bandmate™ Automated Western Blot Processor. Blots were incubated with Thermo Scientific™ Pierce™ Clear Milk Blocking Buffer for 30 minutes. Hsp70 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Primary antibody was removed and GAM-HRP secondary antibody (1:120,000 in TBST) was added to blots and incubated for 2 hours. Thermo Scientific™ SuperSignal™ West Dura chemiluminescent substrate was used for detection. The Invitrogen™ iBright™ FL1500 Imaging System was used for image capture. Local background–corrected volume per lysate was plotted for each lane. (B) 4EBP1: Invitrogen™ Novex™ 16% Tricine mini gels were loaded with 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 μg of A431 lysate per lane, from left to right. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Immunoprocessing was completed using the Bandmate Automated Western Blot Processor. Blots were incubated with Pierce Clear Milk Blocking Buffer for 30 minutes. 4EBP1 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Primary antibody was removed and GAR-HRP secondary antibody (1:175,000 in TBST) was added to blots and incubated for 2 hours. SuperSignal West Dura chemiluminescent substrate was used for detection. The iBright FL1500 instrument was used for image capture. Local background–corrected volume per lysate was plotted for each lane. Note: GAM = goat anti-mouse, HRP = horseradish peroxidase, TBST = Tris-buffered saline with Tween™ solution, GAR = goat anti-rabbit. iBlot 3 system Supplier B semi-dry system Supplier B wet tank system iBlot 3 system Supplier B semi-dry system Supplier B wet tank system 4EBP1 Hsp70 Broad range 25 V, 6 min, and low cooling 25 V, 1.3 A, and 7 min 100 V and 60 min Low molecular weight 25 V, 3 min, and medium cooling 25 V, 1.3 A, and 5 min 100 V and 60 min iBlot 3 system Supplier B semi-dry system Supplier B wet tank system iBlot 3 system Supplier B semi-dry system Supplier B wet tank system 4EBP1 Hsp70 Broad range 25 V, 6 min, and low cooling 25 V, 1.3 A, and 7 min 100 V and 60 min Low molecular weight 25 V, 3 min, and medium cooling 25 V, 1.3 A, and 5 min 100 V and 60 min A B 2,000,000 1,800,000 1,600,000 1,400,000 1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0 5 iBlot 3 Supplier B semi-dry Supplier B wet tank iBlot 3 Supplier B semi-dry Supplier B wet tank A431 lysate (μg) A431 lysate (μg) Local background– corrected volume (intensity) 10 15 20 1,400,000 1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0 2 Local background– corrected volume (intensity) 4 6 8 10 2,000,000 1,800,000 1,600,000 1,400,000 1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0 5 iBlot 3 Supplier B semi-dry Supplier B wet tank iBlot 3 Supplier B semi-dry Supplier B wet tank A431 lysate (μg) A431 lysate (μg) Local background– corrected volume (intensity) 10 15 20 1,400,000 1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0 2 Local background– corrected volume (intensity) 4 6 8 10 62 Western blotting thermofisher.com/proteinbiology Power Blotter systems The Invitrogen™ Power Blotter is our flexible solution for western blot transfer. Designed for rapid 5–10 minute semi-dry transfer of proteins from polyacrylamide gels to nitrocellulose or PVDF membranes, the Power Blotter contains an integrated power supply, LCD touchscreen, and preprogrammed, optimized transfer protocols. Harness the power of the Power Blotter in your western blotting workflow. Highlights: • User-friendly—all-in-one transfer system, with an integrated power supply and touchscreen • Versatile—our most flexible transfer device, with options in throughput, consumables, and transfer speed, designed to grow around your lab’s needs • Efficiency—high transfer efficiency for a broad range of protein sizes (Figure 2) Figure 2. Power Blotter Select Transfer Stacks and Power Blotter Pre-cut Membrane and Filter stacks efficiently transfer high, medium, and low molecular weight proteins. Western blot analysis of KLH, EGFR, Hsp90, PDI, and cyclophilin B was performed by loading serial dilutions of KLH-spiked HeLa lysate with KLH spike (starting at 7.5 μg HeLa lysate, 7.5 μg KLH spike per well, serially diluted 2:3) into Invitrogen™ Bolt™ 4–12% Bis-Tris Plus gels. Proteins were transferred for 7 minutes using a Power Blotter Select Transfer Stack (left) or Power Blotter Pre-cut Membrane and Filter stack (right), and then probed with target-specific primary antibodies and fluorescently conjugated secondary antibodies. Images were captured using automatic exposure on an Invitrogen™ iBright™ FL1000 Imaging System. Power Blotter with Select Transfer Stack Power Blotter with Pre-cut Membrane and Filters and Power Blotter 1-Step Transfer Buffer KLH EGFR Hsp90 PDI EGFR (extra band) Cyclophilin B KLH EGFR Hsp90 PDI EGFR (extra band) Cyclophilin B View the how-to video: Learn more at thermofisher.com/powerblotter Western blotting thermofisher.com/proteinbiology 63 Western blot detection The last step in the western blot workflow, after the separation of proteins by PAGE and their transfer from gel to membrane, is detection. In this step, primary antibodies specific to the protein of interest bind the protein on the membrane. With a variety of detection techniques to choose from (chemiluminescent, fluorescent, or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Quick visualization or precise quantitation, single-probe detection or multiplexing: we offer a range of reagents and instruments for western blot detection and subsequent analysis. Blocking and wash buffers Verified western blot antibodies Sensitive substrates Imaging and analysis Manual vs. automated western blot processing Traditionally, probing a western blot prior to data visualization involves a series of manual steps, many of which are individually short but collectively require significant hands-on time. We offer instruments to automate some of these tasks, tremendously decreasing hands-on time. Automated western blot processing using the Invitrogen™ Bandmate™ Automated Western Blot Processor or Invitrogen™ iBind™ or iBind™ Flex Western Device requires only 15–30 minutes of setup and no additional hands-on steps before you can move to final detection (Figure 3). Primary antibody incubation (1–24 hr) Prepare and load primary antibody, secondary antibody, and wash solutions Steps automated with the Bandmate processor Steps automated with the iBind Devices (3 hr) Secondary antibody incubation (1 hr) 3x wash (5 min each) 5x wash (5 min each) Block Prepare buffers and antibody solutions (20–60 min) Load iBind Card and transfer membrane Prepare and load blocking buffer, wash buffer, and antibody solutions Workflow for traditional manual processing Workflow for iBind Western Devices processing Workflow for Bandmate processor automated processing Figure 3. Comparison of workflows for traditional manual vs. automated western blot processing. The traditional workflow requires more than 10 hands-on steps and at least 4 hours before final detection can occur. Automated processing using the Bandmate Automated Western Blot Processor or iBind or iBind Flex Western Device requires only 15–30 minutes of setup and no additional hands-on steps before final detection. 64 Western blotting thermofisher.com/proteinbiology Bandmate Automated Western Blot Processor The Invitrogen™ Bandmate™ Automated Western Blot Processor is a programmable blot rocking system that automates the tedious hands-on blocking, washing, and antibody incubation steps of western blot processing. Minimal effort is required to set up the Bandmate device to process up to 2 midi blots or 4 mini blots using your current optimized reagents and protocols for blot processing, freeing up time for other important tasks. • Load-and-go setup—prepare block, wash, and antibody solutions, load into the machine, select a program, and walk away • Works with traditional western blotting protocols—no need to switch from current protocols and no specialized reagents required (Figure 4); program the timing of steps based on preference or use preprogrammed options • Antibody recovery—collection tubes can recover antibody for reuse in future experiments if desired Figure 4. Comparison of mini blots processed with the Bandmate Automated Western Blot Processor vs. manual processing (probing and washing steps performed in a tray and on a shaker platform). The blots processed with the Bandmate device show comparable or higher intensity levels across multiple probed targets compared to the blots processed manually. Learn more at thermofisher.com/bandmate View the how-to video: Western blotting thermofisher.com/proteinbiology 65 iBind western detection systems Two devices, one simple technology for western blot processing The Invitrogen™ iBind™ western blot systems are simple, unpowered devices that automate immunodetection steps. The traditional manual process involves preparing and replacing multiple antibody and wash solutions over several hours in a tray containing the blot of interest. In contrast, iBind western systems allow all solutions to be prepared and loaded in the device at the start of the procedure, with subsequent steps proceeding automatically and uninterrupted by sequential lateral flow technology (SLF), i.e., simple capillary action—no electricity or batteries are required. The original iBind Western Device accommodates processing one mini blot at a time, and the iBind Flex Western Device accommodates processing one midi blot, two mini blots, or up to six vertically cut strips at a time for additional flexibility and throughput. Highlights: • Antibody savings—use up to 80% less primary antibody than with traditional tray-based format (Figure 5) • Reproducibility—automated processing provides improved blot-to-blot consistency • Load and go—process solutions using SLF technology, with no batteries, shakers, trays, or timers required GST-tagged proteins (two-fold dilution series) iBind Flex Western System Manual (tray) processing IKK 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 DDR2 FLT1 HCK Figure 5. Comparison of western blots processed manually vs. with the iBind Flex Western Device. Samples containing GST-tagged recombinant proteins were separated on Invitrogen™ NuPAGE™ 4–12%, 20-well midi gels in MOPS SDS running buffer and then transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device. Blots were probed with identical concentrations of the same pair of primary and secondary antibodies. The primary antibody was rabbit anti-GST diluted 1:500 (8 μL in 4 mL iBind Flex Solution for the iBind system, 40 μL in 20 mL for manual tray incubation). The secondary antibody was goat anti–rabbit IgG HRP diluted 1:600 (6.7 μL in 4 mL iBind Flex Solution for the iBind system, 33.3 μL in 20 mL for manual tray incubation). For final detection, blots were incubated for 5 minutes in Thermo Scientific™ SuperSignal™ West Dura Extended Duration Substrate for visualization with an imaging system. Lanes 1–5: IKKβ (80 ng, 40 ng, 20 ng, 10 ng, 5 ng) Lanes 6–10: DDR2 (120 ng, 60 ng, 30 ng, 15 ng, 7.5 ng) Lanes 11–15: FLT1 (40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng) Lanes 16–20: HCK (360 ng, 180 ng, 90 ng, 45 ng, 22.5 ng) Learn more at thermofisher.com/ibind View the how-to video: 66 Western blotting thermofisher.com/proteinbiology Primary antibodies Quality antibodies validated* and cited in many applications The Invitrogen™ antibody portfolio has more than 200,000 antibodies cited across various applications including flow cytometry, IHC, ICC/IF, western blotting, ELISA, IP, and other applications, supporting over 50 research areas. Our primary antibody portfolio offers a broad range of choices with high-quality recombinant monoclonal, monoclonal, and polyclonal antibodies that cover greater than 91% of the proteome. With over 375,000 publications and 835,000 data images and counting, we have the antibodies you can trust. Highlights: • Quality antibodies—continued advanced specificity testing to ensure they bind to the right target (Figure 6) • Purchase with confidence—use our enhanced search experience to filter and compare, so you find the data and information you need • More choices—our broad portfolio allows you to easily find the antibodies you need to support your research Commitment to antibody specificity and reproducibility Thermo Fisher Scientific is working to redefine antibody performance standards with a comprehensive approach to how antibodies are evaluated and validated. Specificity testing is combined with extensive application validation data to provide confidence that our high-quality Invitrogen antibodies will help enable superior performance in your research. Search for antibodies or find out more at thermofisher.com/antibodies A-431 control ~160 kDa ~160 kDa ~45 kDa ~37 kDa p-EGFR EGFR p-MEK1 GAPDH – + A-431 Cas9 – + A-431 EGFR KO – + Figure 6. Western blot analysis using primary antibodies and fluorescent dye–conjugated secondary antibodies. Western blot analysis of phospho-EGFR and phospho-MEK was performed by loading 30 μg of the following whole-cell extracts: A-431 control (lane 1), A-431 treated with EGF (lane 2), A-431 Cas9 (lane 3), A-431 Cas9 treated with EGF (lane 4), A-431 EGFR KO (lane 5), A-431 EGFR KO treated with EGF (lane 6). Phospho-EGFR was detected at ~160 kDa using rabbit polyclonal antibody, EGFR was detected at ~160 kDa using mouse monoclonal antibody, phospho-MEK1 was detected at ~45 kDa using rabbit monoclonal antibody, and GAPDH was detected at ~37 kDa using goat polyclonal antibody. Target proteins were detected using Invitrogen™ Donkey Anti–Mouse IgG Alexa Fluor™ Plus 680 Conjugate, Invitrogen™ Donkey Anti–Rabbit IgG Alexa Fluor™ Plus 800 Conjugate, and Invitrogen™ Donkey Anti–Goat IgG Alexa Fluor™ Plus 555 Conjugate. * The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic use. You can easily identify antibodies that have already undergone this additional testing with the Advanced Verification badge. This badge can be found in antibody search results and on appropriate antibody product web pages. The additional data supporting the Advanced Verification status can be found in the product-specific data image galleries. Advanced Verification Western blotting thermofisher.com/proteinbiology 67 Secondary antibodies Broad menu for multiple detection options Our selection of high-quality secondary antibodies can be used for detection of the primary antibody in a broad range of applications, including western blotting, ICC/IF, flow cytometry, and ELISA. These highly cited, well-characterized secondary antibodies are linked to dyes, enzymes, and labels: including enzyme conjugates, such as HRP and AP, labels such as biotin, and classic fluorescent dyes such as FITC, RPE, and APC. We are also the premier supplier for Invitrogen™ Alexa Fluor™ and Invitrogen™ Alexa Fluor™ Plus dyes. • Fluorescent detection—well suited for studies that need to address levels of protein expression or require simultaneous detection of more than one antigen • Chemiluminescent detection—well suited for low-abundance proteins, and sensitivity can be further improved using our poly-HRP–conjugated secondary antibodies or enhanced chemiluminescent (ECL) substrate to deliver high femtogram–level protein detection • Colorimetric detection—provides quick and easy results and does not require special equipment A B Figure 7. Comparison of detection sensitivity. (A) Invitrogen™ Alexa Fluor™ Plus 800 Secondary Antibody produces more bands at higher dilutions, demonstrating greater sensitivity and range of detection compared to (B) another supplier’s fluorophore-conjugated secondary antibody. 130 kDa 95 kDa 70 kDa 55 kDa 43 kDa Hsp90 HDAC1 130 kDa 95 kDa 70 kDa 55 kDa 43 kDa Hsp90 HDAC1 15.0 μg 5.0 μg 1.6 μg 0.6 μg 0.2 μg 0.06 μg We offer a wide range of antibody conjugates targeting an array of hosts and species derived from a number of sources, including a rich lineup of recombinant secondary antibodies that offer improved sensitivity. Additionally, Thermo Fisher is the only reagent provider offering Invitrogen™ Alexa Fluor™ Plus conjugates (Figure 7). Our extensive menu includes 7 colors and 11 host/target species combinations. Highlights: • Avoid cross-reactivity—use our secondary antibodies that are highly cross-adsorbed against serum proteins or immunoglobulins of other species • Invitrogen™ Alexa Fluor™ and Invitrogen™ Alexa Fluor™ Plus secondary antibodies—get distinct fluorescent bands in multiplex experiments by selecting nonoverlapping spectra for a variety of conjugates using our interactive Fluorescence SpectraViewer tool • Sensitive detection—use Alexa Fluor Plus secondary antibodies or the catalytic activity of our horseradish peroxidase (HRP)-conjugated antibodies in conjunction with SuperBoost™ technology for superior detection of low-abundance proteins Find out more at thermofisher.com/alexafluorplus 68 Western blotting thermofisher.com/proteinbiology Chemiluminescent detection Optimized substrates to enhance sensitivity and reduce background Thermo Scientific™ chemiluminescent HRP substrates offer excellent performance in western blotting applications with longer light emission and stronger signal intensity than other luminol-based detection systems. Choose the appropriate substrate for your needs from the Thermo Scientific™ Pierce™ ECL and Thermo Scientific™ SuperSignal™ families of chemiluminescent HRP substrates. As with other components in a western blotting system, there are many chemiluminescent substrate choices available. The appropriate substrate selection depends on the detection level (sensitivity) required, target protein abundance, and sample availability (Table 2). Highlights: • Excellent sensitivity—substrates providing picogram to attogram sensitivity • Strong light emission—longer signal duration allows for multiple exposures • High intensity—signal is twice as intense as other luminescence-based systems • Antibody savings—our substrates are optimized to work with more dilute primary and secondary antibodies Table 2. Recommended chemiluminescent substrates for western blot detection using HRP. SuperSignal West Dura SuperSignal West Pico PLUS SuperSignal West Atto Choose when Performing quantitative western blotting or maximum signal duration is needed Performing everyday applications; offers improved sensitivity over base-level ECL Target is very low-abundance, sample is limited, or antibodies are limited Features Longest duration and linearity Widest dynamic range Highest sensitivity Detection level Mid-femtogram Low-picogram to femtogram Low-femtogram to high-attogram Signal duration 24 hours Up to 24 hours 6 hours Recommended antibody dilutions (1 mg/mL stock) Primary: 1:5,000 Secondary: 1:50,000–1:250,000 Primary: 1:1,000 Secondary: 1:20,000–1:100,000 Primary: 1:5,000 Secondary: 1:100,000–1:250,000 Western blotting thermofisher.com/proteinbiology 69 iBright Imaging Systems Experience an easier time capturing and analyzing data from gels and western blots with Invitrogen™ iBright™ Imaging Systems. Designed with a streamlined, intuitive interface and workflows, the iBright Imaging Systems family of instruments are easy to use for researchers of all experience levels. There are three models in the iBright Imaging Systems family: the iBright CL750 Imaging System, the iBright CL1500 Imaging System, and the iBright FL1500 Imaging System. The iBright CL750 Imaging System offers the core essential western blot and gel imaging functions and makes the transition from the darkroom and film easy. The iBright CL1500 Imaging System expands application support and has many of the high-performance specifications of our premier iBright FL1500 model. The iBright FL1500 Imaging System features maximum application support, including fluorescent western blot imaging with up to four fluorescence channels at a time. Highlights: • Push-button optimized exposure—Smart Exposure™ acquisition technology for rapid determination of optimal exposure times helps minimize the need to repeat exposures to get the desired signal • Powerful 9.1 megapixel (MP) camera—capture crystal-clear images with robust imaging potential • Advanced automated features—automatic sample rotation,* automatic zoom, automatic focus, and automatic onboard data analysis provide a smooth imaging experience • Five-channel fluorescent blotting—multiplex with the five fluorescence channels of the iBright FL1500 model; capture up to four proteins in a single blot for more meaningful and representative experiments • Compliance support—all models offered with 21 CFR Part 11 compliance support software packages to set up and control security, audit, and e-signature settings iBright Imaging Systems offer up to five imaging modes to support multiple applications (Table 3). Efficiently and easily capture data from protein gels, nucleic acid gels, chemiluminescent western blots, fluorescent western blots, and more (Figure 8). Expanded application support with many of the same high‑performance specifications as the premier iBright FL1500 model Core essential western blot and gel imaging functions to efficiently transition from the darkroom and film Maximum application support, including fluorescent western blot imaging with up to four fluorescence channels at a time Invitrogen™ iBright™ CL750 Imaging System Invitrogen™ iBright™ CL1500 Imaging System Invitrogen™ iBright™ FL1500 Imaging System * Automatic sample rotation available on CL1500 and FL1500 models. 70 Western blotting thermofisher.com/proteinbiology Table 3. Imaging modes available on iBright Imaging Systems. Imaging mode What examples of signal can be captured? Protein gels Colorimetric signals from stains like Thermo Scientific™ Pierce™ Silver Stain or Invitrogen™ SimplyBlue™ SafeStain (gels) and Thermo Scientific™ Pierce™ Reversible Protein Stain or Ponceau S (membranes); fluorescent signals from stains like Invitrogen™ SYPRO™ Ruby Protein Gel Stain (gels). Nucleic acid gels Signals from ethidium bromide and a variety of fluorescent nucleic acid stains like Invitrogen™ SYBR™ stains. Chemi blots Chemiluminescence with horseradish peroxidase (HRP) and alkaline phosphatase (AP) substrates like Thermo Scientific™ SuperSignal™ and Invitrogen™ WesternBreeze™ substrates. Fluorescent blots Fluorescence from visible and near-infrared (NIR) fluorophores like Invitrogen™ Alexa Fluor™ and Alexa Fluor™ Plus conjugates. Universal Custom mode to image with multiple signals (chemiluminescent, fluorescent, colorimetric, and/or visible signals). Image display is similar to display in fluorescent blot mode and allows false colors to be assigned to any sample. View the overview video: Capture your data faster and more easily than ever before. Learn more at thermofisher.com/ibright Test your protein research knowledge Question: Which detection method is most suitable for the detection of more than one antigen on a western blot (i.e., multiplex compatibility)? A. Colorimetric B. Chemiluminescent C. Fluorescent Answer: C Figure 8. Examples of imaging applications. The data were captured in grayscale. Pseudocolor (false color) can be applied for visualization purposes. * Enables qualitative visualization of samples and signal confirmation. Not recommended for quantitation. Colorimetrically stained western blots Fluorescent western blots Fluorescently stained protein gels Chemiluminescent western blots Combined fluorescent and chemiluminescent western blots Colorimetrically stained protein gels Colorimetrically stained membranes Fluorescently stained nucleic acid gels Visible colonies (e.g., stained with crystal violet dye) Fluorescent colonies (e.g., GFP expression) Leaf sections Thin layer chromatography (TLC) plates Core imaging applications Specialty plate–based imaging applications Qualitative visible imaging* applications Western blotting thermofisher.com/proteinbiology 71 Ordering information Product Quantity Cat. No. Protein transfer Mini Gel Tank and Blot Module Set 1 kit NW2000 Mini Blot Module 1 module B1000 SureLock Tandem Midi Welcome Pack, nitrocellulose 1 pack STM4015 SureLock Tandem Midi Welcome Pack, PVDF 1 pack STM4014 SureLock Tandem Midi Blot Module 1 module STM2001 Power Blotter XL System Welcome Pack 1 kit PB0113 Power Blotter System Welcome Pack 1 kit PB0112 Power Blotter XL System 1 system PB0013 Power Blotter System 1 system PB0012 iBlot 3 Starter Kits 1 kit thermofisher.com/ iblotstarter iBlot 3 Western Blot Transfer Device 1 device IB31001 View all available products at thermofisher.com/transfer Automated western blot processing iBind Western Starter Kit 1 kit SLF1000S iBind Western Device 1 device SLF1000 iBind Flex Western Starter Kit 1 kit SLF2000S iBind Flex Western Device 1 device SLF2000 Bandmate Automated Western Blot Processor 1 system BW1000 View all available products at thermofisher.com/detect Blocking buffers StartingBlock (PBS) Blocking Buffer 1 L 37538 StartingBlock (TBS) Blocking Buffer 1 L 37542 Blocker Casein in PBS 1 L 37528 Blocker Casein in TBS 1 L 37532 View all available products at thermofisher.com/westernbuffers Chemiluminescent substrates Pierce ECL Western Blotting Substrate 500 mL 32106 SuperSignal West Pico PLUS Chemiluminescent Substrate 500 mL 34580 SuperSignal West Dura Extended Duration Substrate 100 mL 34075 SuperSignal West Atto Ultimate Sensitivity Substrate 100 mL A38555 View all available products at thermofisher.com/chemisubstrates Antibodies View all available antibodies at thermofisher.com/antibodies Imaging iBright CL750 Imaging System 1 instrument, 1-year warranty, and digital SmartStart Orientation A44116 iBright CL1500 Imaging System 1 instrument, 2-year warranty, and SmartStart Orientation A44240 iBright CL1500 Imaging System 1 instrument, 1-year warranty A44114 iBright CL1500 Imaging System 1 instrument, 2-year warranty, SmartStart Orientation, and license for iBright SAE Software for 21 CFR Part 11 A44240CFR iBright FL1500 Imaging System 1 instrument, 2-year warranty, and SmartStart Orientation A44241 iBright FL1500 Imaging System 1 instrument, 1-year warranty A44115 iBright FL1500 Imaging System 1 instrument, 2-year warranty, SmartStart Orientation, and license for iBright SAE Software for 21 CFR Part 11 A44241CFR View all available models at thermofisher.com/ibright For more information, or to view additional products, go to thermofisher.com/westernblot 72 Western blotting thermofisher.com/proteinbiology Immunoassays Protein assays are an integral part of any laboratory workflow involving protein extraction, purification, labeling, or analysis. Proteins in cell lysates or purified proteins are quantified to verify yield or normalize multiple samples for side-by-side comparison. Popular protein quantitation and characterization techniques include ELISAs and Luminex® assays. Single-biomarker quantitation of protein targets 76 Multiple-biomarker (multiplex) quantitation platforms 81 Ordering information 85 Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Immunoassays thermofisher.com/proteinbiology 73 Immunoassay platforms ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody–antigen interaction. ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from unbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation. A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer). Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic substrates are used most frequently; they allow direct visualization and enable kinetic studies to be performed. Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories. Invitrogen™ ProcartaPlex™ multiplex immunoassays are based on Luminex® xMAP® technology, a bead-based multiplexing technology in which beads are internally labeled with fluorescent dyes to produce a specific spectral address. Instead of attaching capture antibodies to a plate surface, capture antibodies are conjugated to the surface of beads. This technology uses flow cytometric or imaging methods for identification of the beads as well as detection of the bound antigens by the associated reporter molecules labeled with different dyes. The Luminex technology enables multiple proteins to be detected simultaneously in each well of a 96-well plate, using a very small sample volume. Highly referenced kits you can trust To facilitate the investigation of inflammatory and other relevant biological markers, the Invitrogen™ portfolio has a broad menu of immunoassays to easily detect and quantify proteins. Options are available for single- and multi-biomarker quantitation assays, as well as the appropriate instrument platforms for each type of assay, so you can choose the platform you need to publish with confidence. Tailored to meet your needs A variety of assay platforms are available including plate- and bead-based solutions across many different species. ELISA kit formats include complete, ready-to-use kits, as well as preoptimized reagents to make your own. In addition, a variety of singleplex and multiplex panels for protein quantitation with Invitrogen™ ProcartaPlex™ assays are available. See Table 1 for a performance comparison of our immunoassay platforms and Table 2 for a component list. • Invitrogen™ coated ELISA kits—quantitate with confidence. Highly verified ELISA kits with precoated plates provide lower inter- and intra-assay variability with ready-to-use reagents that help ensure consistent data. – Invitrogen™ Instant ELISA™ kits—the one-wash Instant ELISA kit introduces fewer handling steps and requires lower hands-on time to increase productivity. – Invitrogen™ phosphospecific ELISA kits—measure phosphospecific proteins in cell lysates. • Invitrogen™ antibody pair kits—keep costs low with our affordable coat-it-yourself ELISA plate sets. Each set contains the reagents required to prepare and run the ELISA, including ELISA-optimized matched antibody pairs, standards, detection reagents, coating buffers, sample diluent, and tetramethylbenzidine (TMB) substrate solution. Plates are optional. • Invitrogen™ ProQuantum™ high-sensitivity (HS) assay kits—take advantage of a platform innovation that provides researchers with an easy-to-run, high-performance assay, utilizing proximity-based amplification technology to combine the analyte specificity of high-affinity antibody–antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a highly sensitive protein quantitation assay, using very small sample volumes. • ProcartaPlex multiplex immunoassays—quantitate more with less sample. ProcartaPlex multiplex immunoassays utilize Luminex xMAP technology for profiling up to 80 analytes in a single 25–50 μL sample. Biomarker quantitation assay platforms 74 Immunoassays thermofisher.com/proteinbiology Table 2. List of components for different immunoassay kits. Type of immunoassay Immunoassay components Antibody pair ELISA kits Coated ELISA kits Instant ELISA kits ProQuantum HS kits ProcartaPlex assays (all platforms) 96-well plate Optional • • NA Flat bottom Pretitrated capture antibody • Precoated Precoated • Precoated capture bead Pretitrated detection antibody • • • • • Recombinant protein standard • • Additional well strips included • • Stop solution Not included • • NA NA Substrate solution • • • NA SA-PE Assay buffer • • Sample diluent • • Wash buffer Not included • • NA • Coating buffer • NA NA NA NA Note: Our immunoassays are available in various kit sizes. Kits are calibrated to National Institute for Biological Standards and Control (NIBSC) or World Health Organization (WHO) standards (if available). SA = streptavidin, PE = phycoerythrin, NA = not applicable. Go to thermofisher.com/immunoassays to find: • Our helpful immunoassay selection guide that allows you to search for assays based on your target protein • Detailed information on all of our antibody pair kits, ELISA kits, and ProQuantum high-sensitivity and multiplex assay kits • Important data demonstrating our assay specificity and sensitivity Table 1. Characteristics of our immunoassay kits. Type of immunoassay Build-it-yourself immunoassays Uncoated ELISAs and antibody pair kits Coated ELISA kits* Instant ELISA kits ProQuantum HS kits ProcartaPlex immunoassays** Ready-to-use reagents No; need overnight coating process Yes; need overnight coating process Yes Yes Yes Yes Analytical sensitivity† <10 pg/mL <10 pg/mL <10 pg/mL <5 pg/mL <0.1 pg/mL <10 pg/mL Dynamic range† <5–250 pg/mL <5–500 pg/mL <5–250 pg/mL <7.8–500 pg/mL 0.064–5,000 pg/mL <5–2,000 pg/mL Sample volume 10–100 μL 10–100 μL 10–100 μL 25–50 μL 2–5 μL 25–50 μL Incubation time† 4 hr 3–4.5 hr 2.5–4 hr 4 hr 2 hr 3.5 hr Ability to multiplex No No No No No Yes Number of targets measured per well 1 1 1 1 1 1–80 Readout HRP-TMB (colorimetric) HRP-TMB (colorimetric) HRP-TMB (colorimetric) HRP-TMB (colorimetric) FAM™ (fluorescent) RPE (fluorescent) Instrumentation needed Microplate reader Microplate reader Microplate reader Microplate reader RT-PCR Luminex® instrument Instrument read time 2 min 2 min 2 min 2 min 36–72 min 20–60 min * Values in this table refer to standard colorimetric kits. Ultrasensitive kits are also available. ** Immunoassays for the Luminex platform. † Every assay has its own specifications. Please consult the protocol for your specific immunoassays or kits. Immunoassays thermofisher.com/proteinbiology 75 Table 3. Rigorous assay verification of ELISA kits helps ensure consistent, reliable results. Specification Description What does it mean for you? Standard calibration Calibrated to NIBSC, if available Allows accurate quantitation and consistent standard of reference across multiple platforms Precision Average inter-assay CV <10%; average intra-assay CV <10% Consistent results Sensitivity Relevant levels of protein are detected for specific assay type Enables detection of low levels of protein Specificity Cross-reactivity tests are performed with similar analytes Helps to ensure accurate measurement of the protein of interest Recovery Buffers are optimized to minimize matrix effects Helps to ensure accurate quantitation of samples within a complex matrix, including serum and plasma Lot-to-lot consistency In-house controls are tested to measure within set ranges Helps to ensure consistent results with new lots Linearity of dilution Linear results over the quantitative range of the assay Serial dilution of samples are quantitated accurately Parallelism Recombinant protein standards mimic native proteins Samples can be measured with confidence Precoated ELISA kits Our ELISA kits are developed to meet specifications typically expected by customers, including standard calibration, precision, sensitivity, specificity, recovery, lot-to-lot consistency, linearity, and parallelism (Table 3). Coated ELISA kits that include ready-to-use reagents Quantitate with confidence Coated ELISA kits for the quantitation of individual analytes from a variety of sample types provide lower inter- and intra-assay variability. Developed and manufactured for labs that require verified and comprehensively tested kits, coated ELISA kits offer lot-to-lot consistency and help ensure the highest possible ELISA performance (see an example standard curve in Figure 1). Ready-to-use reagents, including precoated plates, help ensure consistent data throughout your research. Highlights: • Robust—low inter- and intra-assay coefficient of variation (CV) • Easy to use—includes all required assay buffers and reagents, and a straightforward protocol • Highly verified—optimized for serum, plasma, and cell culture supernatants Single-biomarker quantitation of protein targets Figure 1. Standard curve for human IgG1 ELISA. Request bulk packaging or lot reservation, or go custom at thermofisher.com/IASpecialServices 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 10 Concentration (ng/mL) Absorption (450 nm) 100 1,000 76 Immunoassays thermofisher.com/proteinbiology Reduce steps and hands-on time by as much as 60% The one-wash Instant ELISA kit requires fewer handling steps than the conventional ELISA kit (Table 4). The Instant ELISA kit contains all of the necessary components, including capture antibody and lyophilized detection antibody, streptavidin-HRP, and sample diluent. Additionally, the strip wells containing the standards for the standard curve are provided separately; they are ready to use, helping save both time and wells. Less hands-on time can help increase productivity, which leaves more time for your research. Instant ELISA: the one-wash ELISA kit Learn more about Instant ELISA kits at thermofisher.com/instantelisa Figure 2. Performance comparison of human sCD44var Instant ELISA to coated ELISA. Human sCD44var Instant ELISA kit was tested against a human sCD44var Coated ELISA kit. A coefficient of determination (R²) of 0.98 was observed using 16 serum samples, demonstrating comparable performance of the Instant ELISA and coated ELISA kits. Table 4. Steps for conventional ELISA vs. Instant ELISA kits. 17 steps for conventional ELISA 7 steps for Instant ELISA kits 1 Washing of coated plate 2 Reconstitution of standard protein 3 Addition of sample diluent to standard wells 4 Titration of standard curve 1 Rehydration of plate 5 Addition of sample diluent 2 Sample addition 6 Sample addition 3 Incubation 7 Dilution of biotin conjugate 8 Addition of biotin conjugate 9 Incubation 10 Preparation of streptavidin- HRP conjugate 11 Washing step 12 Addition of streptavidin- HRP conjugate 13 Incubation 14 Washing step 4 Washing step 15 Addition of TMB substrate 5 Addition of TMB substrate 16 Addition of stop solution 6 Addition of stop solution 17 Calculation of results 7 Calculation of results Highlights: • Time saving—only requires about 15 min for setup • Maximum accuracy—no need to add antibody or perform serial dilution of standards; reduced handling means less error and more consistent results • Better value—generate standard curve data in parallel with additional well strips provided, to enable use of all 96 wells for your samples Switching to Instant ELISA kits The Instant ELISA kits utilize the same antibody pairs for many analytes across all assay formats. This enables cross-platform performance and comparable data using any of our Instant ELISA kits, ELISA, and ProcartaPlex multiplex assays. For example, if you are currently using an Invitrogen™ sCD44var Coated ELISA, you can be confident in switching to the Invitrogen™ sCD44var Instant ELISA™ assay, knowing that the data generated across the two kits will remain consistent (Figure 2). 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 R2 = 0.98 sCD44var Coated ELISA (ng/mL) sCD44var Instant ELISA kits (ng/mL) Immunoassays thermofisher.com/proteinbiology 77 Coat it yourself: optimized reagents, all in one box Uncoated ELISA and antibody pair kits are ideal for experienced ELISA users on a budget, who have less stringent requirements for inter- and intra-assay variance. Affordable ELISA kits using coat-it-yourself plates also include the necessary reagents to prepare and run an ELISA, including ELISA-optimized matched antibody pairs, standards, detection reagents, wash buffers, and coating buffers (plates are optional). Highlights: • Flexible—purchase based on the volume of your assay through a choice of package sizes, including 2-, 10-, and 20-plate kits • Complete and easy to use—includes optimized antibody pairs and recombinant standards (Figure 3), TMB substrate, and all essential reagents and buffers—unlike ELISA kits from other suppliers • Affordable—priced to accommodate the most demanding budgets and to help maximize your research Uncoated ELISA and antibody pair kits Figure 3. Representative standard curve for Invitrogen™ Mouse TGF Beta-1 Uncoated ELISA Kit with Plates. 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 2 Concentration (ng/mL) Absorption (450 nm) 4 6 8 10 12 14 Find out more about uncoated ELISA kits at thermofisher.com/uncoatedelisa 78 Immunoassays thermofisher.com/proteinbiology Choose from over 200,000 quality antibodies, many of them tested for ELISA, as well as buffers, reagents, and accessories to create your own immunoassay. Stand-alone products enable full flexibility to build an assay to meet your research needs. Thermo Fisher Scientific offers a comprehensive line of accessory reagents to support your immunoassay needs. Reagents include: • Coated and uncoated microplates • Blocking buffers • Diluents and wash buffers • Substrates • Primary and secondary antibodies • Matched antibody pairs Use our broad primary antibody portfolio to design your own ELISA. An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two different nonoverlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are called matched pairs and are suitable for developing a sandwich ELISA. Use our Antigen Search tool to find antibodies to compatible binding regions. With our unique search experience, you can quickly identify the most suitable antibodies from our broad catalog. Search for antibodies or find out more at thermofisher.com/antibodies Build-it-yourself reagents See a complete listing of accessory reagents for ELISA at thermofisher.com/elisaaccessories Find your optimal set of ELISA reagents using the ELISA Builder ELISA Builder makes it easy to find the best solution for developing your own ELISA assay. Based on a short list of questions about your experimental needs, the ELISA Configurator tool recommends everything needed to perform an ELISA, from plates to stop solution. Learn more at thermofisher.com/elisabuilder Immunoassays thermofisher.com/proteinbiology 79 The ProQuantum high-sensitivity immunoassay is a platform innovation that provides researchers with an easy-to-run, high-performance assay with no proprietary instrument to purchase. By utilizing proximity-based amplification technology, we have combined the analyte specificity of high-affinity antibody–antigen binding with the signal detection capabilities of real-time PCR, to achieve a highly sensitive protein quantitation assay with a large dynamic range. With its ability to detect even lower levels of protein than traditional methods with very small sample consumption, you can get the most out of your precious or limited samples. Here’s how it works The ProQuantum immunoassays utilize a matched pair of target-specific antibodies each conjugated to a DNA oligonucleotide. During antibody–analyte binding, the two oligos are brought into close proximity, which then allows for ligation of the two strands and subsequent creation of a template strand for amplification. This proximity-based technology leverages the sensitivity and large dynamic range of Applied Biosystems™ TaqMan™ real-time PCR technology (Figure 4). ProQuantum high-sensitivity immunoassay kits Highlights: • High-sensitivity—detect low levels of proteins with greater sensitivity than with traditional ELISA methods • Broad dynamic range—≥5 orders of magnitude, minimizing sample dilutions to help ensure they fall within the range (Figure 5) • Small sample consumption—use 2–5 μL of sample (for example, 2 μL vs. 75 μL for triplicate wells with other methods) • Fast, easy workflow—2 hr from sample to answer, no wash steps, and a single 1 hr incubation • No proprietary instrument to purchase—runs on any real-time PCR instrument • Includes intuitive analysis software— comprehensive data analysis and statistical groupwise comparison Figure 5. Superior sensitivity and broader dynamic range. Standard curves for Invitrogen™ ProQuantum™ High-Sensitivity Human IFNγ, IL-8, IL-1β, and IL-6 immunoassay kits (red) show larger dynamic range and improved separation at the low end of the curve for greater sensitivity than normal and ultrasensitive ELISAs (purple). 0 2 4 6 8 10 12 14 16 0.125 0.25 0.5 1 2 4 Ct A450 A450 0 2 4 6 8 10 12 14 16 0.0625 0.125 0.25 0.5 1 2 4 IL-6, pg/mL IL-8, pg/mL IL-1, pg/mL IFN , pg/mL 0 2 4 6 8 10 12 14 16 0.0625 0.125 0.25 0.5 1 2 4 Ct Ct A450 Ct A450 0.01 0.1 1 10 100 1,000 10,000 0.01 0.1 1 10 100 1,000 10,000 0.01 0.1 1 10 100 1,000 10,000 0.01 0.1 1 10 100 1,000 10,000 0 2 4 6 8 10 12 14 16 0.125 0.25 0.5 1 2 4 Figure 4. How ProQuantum immunoassay works. (A) Antibody conjugates binding to target (1 hr incubation). (B) Ligation and amplification of signal (in qPCR instrument). 3´ oligo Target Target A B Target 5´ oligo 5´ 5´ 3´ 3´ Fluorescence Cycle number Ct Protein conc. 5PL regression A B Find out more and select your assay at thermofisher.com/proquantum 80 Immunoassays thermofisher.com/proteinbiology Protein multiplex immunoassays Quantitate up to 80 analytes in only 25 μL of serum or plasma, or 50 μL of cell culture supernatant with ProcartaPlex multiplex assays. Gene expression multiplex assays Measure up to 80 RNA transcripts in a single well with Invitrogen™ QuantiGene™ Plex assays. Luminex instruments for target quantitation The Luminex xMAP technology combines advanced fluidics, optics, and digital signal processing with fluorescently dyed microspheres to enable the quantitation of multiple nucleic acid or protein targets from a single sample. ProcartaPlex multiplex immunoassays are designed for measurement of protein expression, and QuantiGene Plex assays are available for nucleic acid quantitation (Table 5). High-throughput immunoassays and gene expression analysis—all from one supplier, on one instrument As a partner of Luminex, Thermo Fisher Scientific has been providing Luminex platform users with a comprehensive offering of instruments and multiplex reagents for over 20 years. ProcartaPlex and QuantiGene Plex assays are available in 96-well and 384-well formats for high-throughput analysis. Multiple-biomarker (multiplex) quantitation platforms ProcartaPlex multiplex immunoassays ProcartaPlex multiplex immunoassays are bead-based assays for protein detection using Luminex xMAP technology. ProcartaPlex assays are based on the principles of a sandwich ELISA, using two highly specific antibodies binding to different epitopes of one protein. Invitrogen™ ProcartaPlex™ Simplex kits and panels can be easily combined to create your own panels, or we can mix and validate a panel for you. Why use Luminex technology? Profile more biomarkers with less starting sample: quantitate up to 80 analytes simultaneously with the INTELLIFLEX™, FLEXMAP 3D®, or Luminex® 200™ instrument, and up to 50 analytes with the MAGPIX® instrument. All of our ProcartaPlex assays are compatible with as little as 25 μL of plasma or serum, or 50 μL of cell culture supernatant. Multiomics Perform multiomics studies using a proven technology on a trusted platform. Hundreds of peer-reviewed publications have cited ProcartaPlex and QuantiGene Plex assays. You can have the advantage of looking at both genomics and proteomics all on one validated instrument platform. To learn more about QuantiGene assays, visit thermofisher.com/quantigene. Table 5. Comparison of different multiplex assay platforms. Assays Analyte measured Plex level Targets available Custom designs ProcartaPlex multiplex immunoassays Proteins 80 >500 Yes QuantiGene Plex RNA assays RNA 80 >17,000 Yes Immunoassays thermofisher.com/proteinbiology 81 Quick, cost-effective, and accurate multi-analyte profiling system The open-architecture of Luminex xMAP technology uses flow cytometry, microspheres, lasers, digital signal processing, and traditional chemistry—combining proven technologies in a unique way. Benefits: • Helps reduce costs and labor through multiplexing • Smaller sample size requirements compared to single-result assays • Enables fast, reproducible results from favorable kinetics of liquid bead array approach • Broad coverage of applications, including protein expression and gene expression, to satisfy the need to understand both proteomics and genomics profiles How Luminex xMAP technology works Sets of magnetic microspheres are color-coded with combinations of red and infrared fluorophores. Each bead set is coated with a reagent specific to a particular biomarker, allowing the capture and detection of several specific biomarkers from a single sample. Lasers in the compact analyzer excite the internal dyes that identify each microsphere as well as any reporter dye captured, providing quantitation of individual analytes on each microsphere. The analyzer reads many beads from each set, enabling rapid and precise results for several targets within a single sample. Luminex xMAP technology 6.5 microns The bead is impregnated with the dye mixture Bead set 26 Bead set 21 10 unique infrared dye concentrations 10 unique red dye concentrations Luminex xMAP technology ProcartaPlex assays have adopted the Luminex xMAP magnetic bead technology that uses Luminex® MagPlex® superparamagnetic 6.5-micron microsphere beads with a magnetic core. The beads are internally dyed with precise proportions of red and infrared fluorophores. Varying proportions of the red and infrared fluorophores result in 100 spectrally unique microspheres, which are identified by the Luminex xMAP detection systems. The conjugation of a distinct monoclonal antibody to a distinct bead allows for analysis of multiple analytes in a single well. View the how-to video: Watch our videos at thermofisher.com/immunoassayvideos to learn how to use our ProcartaPlex products. 82 Immunoassays thermofisher.com/proteinbiology Profile more biomarkers with less sample ProcartaPlex multiplex immunoassays use the Luminex xMAP (multi-analyte profiling) technology that enables the simultaneous detection and quantitation of up to 80 protein targets in a single 25–50 μL sample—from plasma, serum, cell culture supernatants, and other bodily fluids. The technology employs the use of differentially dyed capture beads for each target in a multiplex ELISA-like assay. ProcartaPlex immunoassays can profile up to 80 times more analytes using significantly less sample in the same time that it takes to perform a traditional sandwich ELISA. ProcartaPlex multiplex immunoassays are available in off-the-shelf kits or you can create your own unique panel through our mix-and-match panel offering (Table 6). It is often advantageous to start with a large panel and then reduce the number of biomarkers analyzed in a more focused panel. Whatever option works best for you, ProcartaPlex panels enable reproducible results throughout the course of your study. ProcartaPlex multiplex immunoassay kits • More than 90% of ProcartaPlex assays can be combined with one another • All ProcartaPlex assays are individually tested for specificity and endogenous, native protein detection • Multi-analyte detection with a large dynamic range • Scalable and reproducible performance regardless of plex size • Largest multiplex panel on the market for quantitation of up to 80 analytes ProcartaPlex multiplex immunoassays are available in multiple formats across six species (human, mouse, rat, nonhuman primate, porcine, and canine) to meet the needs of your research. Get information regarding characteristic assay details and individual analytes at thermofisher.com/procartaplex Table 6. ProcartaPlex multiplex assay options. Name Description Mixing required Bead type Base kit included ProcartaPlex Preconfigured panels Predefined, biologically relevant, and disease-defined panels using magnetic beads for the quantitative multiplex analysis of up to 80 analytes in a single sample. Optimal performance and reproducibility through extensive testing for combinability, cross-reactivity, and interference. No Magnetic Yes ProcartaPlex Mix & Match panels Custom-blended and optimized panels deliver results tailored to the panel design of your choice. Simply provide your desired species, sample type, and instrument for use. Then select your desired analytes, and we will build and optimize a custom assay kit according to your specifications. No Magnetic Yes ProcartaPlex Simplex sets Bead sets for the detection of individual analytes designed to be added to ProcartaPlex panels for increased customization. Alternatively, multiple ProcartaPlex simplex bead sets can be combined and run using the Invitrogen™ ProcartaPlex™ Basic Kit, which includes all non–target-specific reagents needed to perform the assay. Yes Magnetic No Have technical questions? Need help getting started? Email luminexfas@thermofisher.com to get a one-on-one technical consultation. Immunoassays thermofisher.com/proteinbiology 83 Table 7. Comparison of Luminex systems for multiplexing. Instrument Luminex xMAP INTELLIFLEX DR-SE System Luminex xMAP INTELLIFLEX System Luminex FLEXMAP 3D Instrument System Luminex 200 Instrument System Most advanced Luminex platform Higher-throughput multiplexing Most established platform Applications Protein and nucleic acid analysis Multiplex capacity Up to 500 targets Up to 100 targets (80 on MagPlex microspheres) Max panel size 80 biomarkers Sensitivity 0.06–1 pg/mL (immunoassays; assay and target dependent); 1,000 RNA copies per target/well (RNA assays) Dynamic range ≥5.5 orders of magnitude (RP1) ≥4.5 orders of magnitude (RP2) ≥5.5 orders of magnitude (RP1) ≥4.5 orders of magnitude (RP1) ≥3.5 orders of magnitude (RP1) Sample volume (assay dependent) 6.3–80 μL 25–80 μL Plate format 96-well 384-well 96-well Read time 20 min (96-well) 75 min (384-well) 45 min (96-well) Footprint 58.4 x 61 x 76 cm 110 x 62 x 63 cm 115 x 60 x 50 cm Optics/hardware Flow cytometry–based lasers, APDs, PMTs Reporter laser/optics 532 nm (green) and 405 nm (violet) 532 nm (green) 532 nm (green) 532 nm (green) Operational validation/ compliance Calibration, verification, and fluidics 21 CFR compliance (coming in 2022 for INTELLIFLEX instrument) Automation Automation integration software available (coming in 2022 for INTELLIFLEX instrument) Analytics (software) Data analysis apps for Luminex assays on the Thermo Fisher™ Connect Platform xPONENT™ basic plus partner analytics The Luminex family of instruments includes options for various multiplexing capabilities, throughput, and read times (Table 7). All instruments are capable of protein and nucleic acid applications, and all are supported by commercially available assays that can multiplex up to 80 biomarkers simultaneously. Luminex instrumentation Accessories to keep your Luminex instruments running strong include sheath fluid, calibrator and control microspheres, verification and calibration kits, and hand-held plate washers. Learn more at thermofisher.com/luminexinstruments Test your protein research knowledge Question: Chromogenic ELISA assays (select all that are true): A. Are the most commonly used ELISA detection method B. Enable kinetic studies C. Are as sensitive as chemiluminescent ELISAs D. Use a standard absorbance plate reader Answer: A, B, and D 84 Immunoassays thermofisher.com/proteinbiology Ordering information Product Quantity Cat. No. ELISA kits Amyloid Beta 40 ELISA Kit, Human 96 assays KHB3481 192 assays KHB3482 Amyloid Beta 42 ELISA Kit, Human 96 assays KHB3441 192 assays KHB3442 Amyloid Beta 42 ELISA Kit, Mouse 96 assays KMB3441 Amyloid Beta 42 Ultrasensitive ELISA Kit, Human 96 assays KHB3544 Tau (Total) ELISA Kit, Human 96 assays KHB0041 192 assays KHB0042 Tau [pS396] Phospho-ELISA Kit, Human 96 assays KHB7031 Tau [pT181] Phospho-ELISA Kit, Human 96 assays KHO0631 AMPK Alpha-1,2 [pT172] Phospho-ELISA Kit, Human 96 assays KHO0651 STAT3 [pY705] Phospho-ELISA Kit, Multispecies 96 assays KHO0481 sIL-2R/CD25 (Soluble) ELISA Kit, Human 96 assays BMS212-2 IL-6 ELISA Kit, Human 96 assays KHC0061 96 assays KAC1261 96 assays EH2IL6 IL-6 ELISA Kit, Mouse 96 assays KMC0061 IL-8 ELISA Kit, Human 96 assays KHC0081 IL-10 ELISA Kit, Human 96 assays KAC1321 IFN Beta ELISA Kit, Human 96 assays 414101 IFN Alpha ELISA Kit, Human 96 assays BMS216 IFN Gamma ELISA Kit, Human 96 assays KAC1231 TGF Beta-1 ELISA Kit, Human 96 assays BMS249-4 TNF Alpha ELISA Kit, Human 96 assays KHC3011 96 assays KAC1751 TNF Alpha ELISA Kit, Mouse 96 assays BMS607-3 192 assays BMS607-3TWO SAA ELISA Kit, Human 480 assays KHA0011C SAA ELISA Kit, Mouse 96 assays KMA0021 SAA ELISA Kit, Livestock 96 assays KAA0021 Insulin ELISA Kit, Human 96 assays KAQ1251 Leptin ELISA Kit, Human 96 assays KAC2281 Adiponectin ELISA Kit, Human 96 assays KHP0041 Alpha-Synuclein ELISA Kit, Human 96 assays KHB0061 VEGF ELISA Kit, Human 96 assays KHG0111 192 assays KHG0112 c-Myc (Total) ELISA Kit, Human 96 assays KHO2041 IgG Subclass ELISA Kit, Human 192 assays 991000 Rapid ELISA Mouse mAb Isotyping Kit 60 tests 37503 To view additional products, go to thermofisher.com/ELISA Immunoassays thermofisher.com/proteinbiology 85 Product Quantity Cat. No. ProcartaPlex panels Human Immune Monitoring 65-Plex Human ProcartaPlex Panel 96 tests EPX650-10065-901 Cytokine/Chemokine/Growth Factor Convenience 45-Plex Human Panel 1 96 tests EPXR450-12171-901 Cytokine & Chemokine Convenience 34-Plex Human Panel 1A 96 tests EPXR340-12167-901 Cytokine Storm 21-Plex Human Panel 96 tests EPX210-15850-901 Inflammation 20-Plex Human Panel 96 tests EPX200-12185-901 Immuno-Oncology Checkpoint 14-Plex Human Panel 1 96 tests EPX14A-15803-901 Coronavirus Ig Total Human 11-Plex Panel 96 tests EPX110-16000-901 SARS-CoV-2 Variants Neutralizing Antibody 6-Plex Panel 96 tests EPX060-16018-901 Mouse Immune Monitoring 48-Plex Mouse Panel 96 tests EPX480-20834-901 Cytokine & Chemokine Convenience 36-Plex Mouse Panel 1A 96 tests EPXR360-26092-901 Immuno-Oncology Checkpoint 7-Plex Mouse Panel 2 96 tests EPX070-20835-901 Rat Antibody Isotyping 6-Plex Rat Panel 96 tests EPX060-30123-901 Kidney Toxicity 5-Plex Rat Panel 1 96 tests EPX050-30124-901 Canine Cytokine/Chemokine/Growth Factor 11-Plex Canine Panel 1 96 tests EPX11A-50511-901 Porcine Cytokine & Chemokine 9-Plex Porcine Panel 1 96 tests EPX090-60829-901 Nonhuman primate (NHP) Cytokine/Chemokine/Growth Factor 37-Plex NHP Panel 96 tests EPX370-40045-901 Cytokine & Chemokine 30-Plex NHP Panel 96 tests EPX300-40044-901 To view additional products, go to thermofisher.com/luminex ProQuantum panels IL-6 Human ProQuantum Immunoassay Kit 96 tests A35573 TNF Alpha Human ProQuantum Immunoassay Kit 96 tests A35601 IL-8 Human ProQuantum Immunoassay Kit 96 tests A35575 IFN Alpha Human ProQuantum Immunoassay Kit 96 tests A42897 TNF Alpha Mouse ProQuantum Immunoassay Kit 96 tests A43658 Mouse IL-17A ProQuantum Immunoassay Kit 96 tests A46737 To view additional products, go to thermofisher.com/proquantum 86 Immunoassays thermofisher.com/proteinbiology Tandem Mass Tag (TMT and TMTpro) systems 90 SureQuant assays and standards 92 HeavyPeptide and LightPeptide AQUA custom standards 93 Protein sample preparation 95 Instrument calibration 97 Ordering information 99 Mass spectrometry Mass spectrometry (mass spec or MS) has become a method of choice for protein analysis. The accuracy, sensitivity, and flexibility of MS instruments have enabled new applications in biological research, biopharmaceutical characterization, and diagnostic detection. MS can identify and quantify known and unknown compounds by revealing their structural and chemical properties. With its many forms of ionization and measurement, MS enables the analysis of samples ranging in mass from 50 to 300,000 Da, in attomole through nanomole quantities. Proper sample preparation and chromatography, as well as the right instrumentation and software, are critical components of successful MS-based proteomic analysis. Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Mass spectrometry thermofisher.com/proteinbiology 87 Protein quantitation using mass spectrometry Differences in protein expression can be studied both globally (discovery proteomics) or within a specific subset of proteins (targeted proteomics). Most quantitative proteomic analyses utilize the isotopic labeling of proteins or peptides in the experimental groups, which can then be differentiated by mass spectrometry. Relative quantitation methods are used to compare protein or peptide abundance between samples, while spiking unlabeled samples with known concentrations of isotopically labeled synthetic peptides can help to enable absolute quantitation of target peptides via selected reaction monitoring (SRM). Discovery quantitation strategies for relative protein quantitation include stable isotope labeling using amino acids in cell culture (SILAC) and labeling using tandem mass tag (TMT) reagents. SILAC-based quantitation involves metabolically labeling protein samples in cultured mammalian cells with a heavy isotope– labeled form of an amino acid. Inclusion of the labeled amino acid in cell or tissue culture media results in replacement of the natural light amino acid with the heavy form in newly synthesized proteins, enabling multiplexing. Isobaric chemical tags are a more universal alternative to SILAC because they can be used with a wide variety of samples including cells, tissues, and biological fluids. Isobaric chemical tags facilitate the simultaneous analysis of up to eighteen samples and consist of an MS/MS reporter group, a spacer arm, and a reactive group. Amine-reactive groups covalently bind to peptide N termini or to lysine residues. Each tag fragments during MS/MS, producing unique reporter ions, enabling multiplexed quantitation through comparing the intensities of the reporter ions. Absolute quantitation is performed in targeted proteomic experiments and increases the sensitivity of detection for a limited number of target analytes. These approaches require spiking a sample with known amounts of synthetic peptides containing heavy stable isotopes, which act as internal quantitative standards for absolute quantitation of the corresponding natural peptides in the sample. Sample preparation Sample preparation is one of the most variable and timeconsuming steps in the analysis of proteins by MS, and the quality and reproducibility of sample extraction and preparation significantly impact the results. Robust, integrated workflows enable consistent results between labs and help eliminate wasted time spent troubleshooting experimental methods and results. Protein extraction, depletion, and enrichment strategies have been developed to remove high-abundance proteins or isolate target proteins in the sample, reduce sample complexity, and help improve the detection of low-abundance proteins. Digestion is required because proteins are often too big and complex for analysis and therefore are digested into peptides for MS analysis for better detection and identification of proteins. Trypsin is the protease of choice for protein digestion. However, digestion with alternative proteases such as Glu-C, Lys-N, Lys-C, Asp-N, or chymotrypsin can improve individual protein sequence coverage or generate unique peptide sequences for different MS applications. Instrument calibration Calibration solutions and standards are critical for optimal performance in mass spectrometry; mass spectrometers must be carefully monitored to ensure accuracy of results. Ideally, calibration reagents should be ready-to-use liquid formulations composed of highly purified, ionizable molecules or polymers specifically designed for positive or negative calibration of instruments. Standards should be designed for specific applications, including sensitivity assessment, determination of digestion efficiency, chromatography assessment, or as controls for sample analysis. 88 Mass spectrometry thermofisher.com/proteinbiology Protein quantitation We offer reagents and kits for both discovery and targeted proteomics applications (Table 1). Discovery proteomics utilizes techniques such as SILAC and isobaric mass tagging that allow the user to look at global protein expression levels. In contrast, targeted proteomics utilizes selected or multiple reaction monitoring (SRM/MRM), which are methods of absolute quantitation (also termed AQUA), in which a complex sample is spiked with a stable isotope-labeled peptide that acts as internal standard for a specific subset of proteins. Table 1. Choose the right protein quantitation reagent for mass spectrometry. SILAC TMT TMTpro Peptides for SRM SureQuant targeted assays Application type Discovery quantitation Targeted quantitation Labeling method Metabolic Amine-, cysteine-, and carbonylreactive mass tags Amine-reactive mass tags Spike-in standard Spike-in standard Sample type Cultured mammalian cells Cultured mammalian cells; tissue; biofluids Cultured mammalian cells; tissue; biofluids Cultured mammalian cells; tissue; biofluids Cultured mammalian cells; tissue; biofluids Digestion protocol In-gel In solution In solution In solution In solution Quantitation mode MS1 MS2 (6-plex) MS3 (10-plex) MS3 (11-plex) MS3 (16-plex) MS3 (18-plex) MS1 MS1 Multiplex options 2-plex, 3-plex, 4-plex 2-plex, 6-plex, 10-plex, 11-plex 16-plex, 18-plex Samples not combined Samples not combined Mass spectrometry thermofisher.com/proteinbiology 89 N NH O N O O O O 126 * * * * * N NH O N O O O O * 127N * * * * N NH O N O O O O * * * * * 129N N NH O N O O O * O * * * * 130C N NH O N O O O * O * * * * 128C N NH O N O O O * O * * * 131 * A. TMT10plex Reagents (TMT10) N NH O N O O O O * * * * * 127C N NH O N O O O O * * 128N * * * N NH O N O O O O * * * * * 129C N NH O N O O O * O * 130N * * * A. TMT Reagent Generic Chemical Structure N NH O N O O O O Mass Reporter Mass Normalizer Amine-Reactive Group HCD ETD N NH O N O O O O 126 * * * * * N NH O N O O O O * 127 * * * * N NH O N O O O O * * * * * 129 N NH O N O O O * O * * * * 130 N NH O N O O O * O * 128 * * * N NH O N O O O * O * * * 131 * B. TMTsixplex Reagents (TMT6) Simultaneously identify and quantify protein expression from multiple samples in a single analysis The Thermo Scientific™ TMT™ and TMTpro™ Label Reagent Sets enable simultaneous quantitation of up to 11 (TMT reagents) or 18 (TMTpro reagents) samples, providing the highest throughput available for protein identification and quantitative analysis by tandem mass spectrometry (MS). Highlights: • Multiplex—concurrent MS analysis of up to 11–18 samples derived from cells, tissues, or biological fluids (Figures 1–3) • Robust—increased multiplex capability results in fewer missing quantitative values among samples and higher confidence among replicates • Efficient—amine-reactive, NHS ester–activated reagents ensure efficient labeling of all peptides regardless of protein sequence or proteolytic enzyme specificity • Convenient—provided in a ready-to-use, single-use format to ensure optimal stability and performance or in bulk for custom formatting Tandem Mass Tag (TMT and TMTpro) systems A B Figure 1. Chemical structures of TMT reagents. (A) Structures of TMT10plex reagents with ¹³C and ¹⁵N heavy-isotope positions (red asterisks). (B) Structures of TMTsixplex reagents with ¹3C and ¹5N heavy-isotope positions (red asterisks). 90 Mass spectrometry thermofisher.com/proteinbiology Mass reporter Mass normalizer Cleavable linker TMTpro-126 TMTpro-127C TMTpro-128C TMTpro-129C TMTpro-130C TMTpro-131C TMTpro-132C TMTpro-133C TMTpro-127N TMTpro-128N TMTpro-129N TMTpro-130N TMTpro-131N TMTpro-132N TMTpro-133N TMTpro-134N Amine-reactive group Figure 2. Structures of TMTpro 18plex reagents with ¹3C and ¹5N heavy-isotope positions (red asterisks). Figure 3. Procedure summary for MS experiments using TMTpro isobaric mass tagging reagents. Protein extracts isolated from cells or tissues are reduced, alkylated, and then digested using the Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit or an equivalent method. Samples are then labeled with the TMTpro reagents before sample mixing, fractionation, and cleanup. Labeled samples are analyzed on a high-resolution Thermo Scientific™ Orbitrap™ LC-MS/MS mass spectrometer before data analysis to identify peptides and quantify relative abundance of reporter ions. TMTpro 126 TMTpro 127N : : : TMTpro 134N Label Sample 1 Sample 2 : : : Sample 16 Treat samples and isolate proteins Denature, reduce, alkylate, do tryptic digest Fractionate, clean up, perform LC-MS/MS analysis Data analysis and quantitation using Proteome Discoverer software Combine Learn more at thermofisher.com/tmtpro Mass spectrometry thermofisher.com/proteinbiology 91 Validated, modular reagents for multiplexed targeted protein quantitation The Thermo Scientific™ SureQuant™ assays and standards have been designed for multiplexed targeted quantitation for key cellular signaling proteins. The assays provide flexibility to choose the use of the immunoprecipitation sample prep kits and peptide quantitation modules together or independently to best suit the end application. The MS sample preparation has been streamlined to be completed in ~4 hours. Together, SureQuant assays and standards provide a complete solution for multiplex IP enrichment and analysis of target proteins (Figures 4 and 5). SureQuant assays and standards Highlights: • Complete—includes reagents for successful sample preparation and quantitative analysis of each target peptide • Verified—kits and reagents rigorously tested for specificity and successful quantitation of each target peptide • Multiplex—Thermo Scientific™ HeavyPeptide™ AQUA panels for simultaneous quantitation of target proteins and phosphorylation status from key cell signaling pathways • Flexible—modular format allows for immunoenrichment only, or in combination peptide quantitation panels Learn more at thermofisher.com/ms-targeted-assays Figure 4. Multiplex IP analysis using streptavidin magnetic beads with human tissue. AKT/mTOR pathway proteins were enriched through multiplex IP using biotinylated antibodies from Thermo Scientific™ SureQuant™ AKT pathway kits. Parallel reaction monitoring (PRM) analysis for (A) total AKT/mTOR pathway targets and (B) phosphorylated AKT/mTOR pathway targets was performed using the Thermo Scientific™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. Data were subsequently analyzed in Skyline software using calibration curves to determine the absolute protein concentration (fmol). Figure 5. Use of the Thermo Scientific™ SureQuant™ Multipathway Phosphopeptide Standard with phosphopeptide enrichment enables relative quantitation of greater than 100 phosphoproteins. Heat map of relative levels of phosphopeptides (left). Changes in specific phosphopeptides (right). One microgram of treated cell lines was incubated with Fe-NTA magnetic beads. Prior to enrichment, 1 pmol of the SureQuant Multipathway Phosphopeptide Standard was spiked into the lysate. PRM analysis was performed using the UltiMate 3000 RSLCnano System and Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer and subsequently analyzed in Skyline software using the average total area. A: HCT116/IGF; B: MCF7/IGF; C: LNCaP/IGF; D: A431/EGF; E: HepG2/Ins. 0 10 20 30 50 40 70 60 90 80 AKT1 AKT2 AKT3 AKT1S1 GSK3 GSK3AKT1 AKT2 AKT3 AKT1S1 GSK3 GSK3 IGF1R IRS1 mTOR PTEN KS6B1 TSC2  IGF1R IRS1 mTOR PTEN KS6B1 TSC2 fmol target Breast normal Breast tumor Large intestine normal Large intestine tumor 0 2 4 6 8 10 12 fmol target 0 10 20 30 50 40 70 60 90 80 AKT1 AKT2 AKT3 AKT1S1 GSK3 GSK3 IGF1R IRS1 mTOR PTEN KS6B1 TSC2 AKT1 AKT2 AKT3 AKT1S1 GSK3 GSK3 IGF1R IRS1 mTOR PTEN KS6B1 TSC2 fmol target Breast normal Breast tumor Large intestine normal Large intestine tumor 0 2 4 6 8 10 12 fmol target AKT1:S473 FOXO3:S294 2.0 x 107 0.0 x 107 4.0 x 107 6.0 x 107 8.0 x 107 1.2 x 108 1.0 x 108 1.4 x 108 1.6 x 108 1.0 x 106 0.0 x 106 2.0 x 106 3.0 x 106 4.0 x 106 6.0 x 106 5.0 x 106 7.0 x 106 8.0 x 106 5.0 x 106 0.0 x 106 1.0 x 107 1.5 x 107 2.0 x 107 3.0 x 107 2.5 x 107 3.5 x 107 1.0 x 107 0.0 x 107 2.0 x 107 3.0 x 107 4.0 x 107 5.0 x 107 6.0 x 107 2.0 x 105 0.0 x 105 4.0 x 105 6.0 x 105 8.0 x 105 1.0 x 106 1.2 x 106 5.0 x 106 0.0 x 106 1.0 x 107 1.5 x 107 2.0 x 107 2.5 x 107 A B C D E STAT3:Y705 IRS1:S1101 STAT1:Y701 TSC2:S939 A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E Row Z-Score −1 0 +1 89 31 115 23 35 23 128 4 119 91 41 95 104 103 5 83 60 65 13 38 12 78 87 110 37 100 56 49 86 52 85 54 81 129 127 30 123 59 84 75 71 74 10 11 107 106 46 130 21 72 66 33 32 39 9 28 17 99 16 87 50 22 27 111 64 62 25 69 73 122 93 67 88 114 34 125 118 109 96 36 48 105 43 55 57 3 131 20 19 126 92 79 53 120 82 108 58 40 51 15 76 112 77 68 124 18 121 29 90 63 101 94 6 116 1 45 117 113 102 14 61 70 42 47 80 24 98 97 2 44 A B 92 Mass spectrometry thermofisher.com/proteinbiology Improve quantitative precision for absolute quantitation The Thermo Scientific™ HeavyPeptide™ and LightPeptide™ AQUA custom synthesis services provide isotopically labeled or unlabeled AQUA (Absolute QUAntitation) peptides with extensive QC for relative and absolute quantitation of proteins. HeavyPeptide and LightPeptide standards are synthesized using the latest Fmoc solid-phase peptide synthesis technology, purified by HPLC, and analyzed by mass spectrometry. Amino acid analysis (AAA) provides the most exact peptide concentration to guarantee the highest level of quantitative precision. The different grades of AQUA peptides are defined by the replicates of AAA, with Basic (1X), QuantPro (2X), and Ultimate (3X). Highlights: • Precise—peptide concentration guaranteed from AAA • Sensitive—enables absolute quantitation of low‑abundance proteins (fmol) • Specific—100% peptide sequence specificity • Flexible—variety of purity, modification, and formatting options (Table 2) HeavyPeptide and LightPeptide AQUA custom standards Table 2. Specifications of HeavyPeptide AQUA-grade standards. AQUA Ultimate Service AQUA QuantPro Service AQUA Basic Service Research stage Validation, ideal for absolute quantitation Confirmation or validation, ideal for biomarker verification Discovery, confirmation or validation, relative quantitation Amount/number of aliquots 10 nmol/10 aliquots 40 nmol/40 aliquots 96 nmol/96 aliquots 10 nmol/10 aliquots 40 nmol/40 aliquots 96 nmol/96 aliquots 15 to 30 nmol (0.05 to 0.1 mg)/1 aliquot Formulation 5 pmol/μL in 5% (v/v) acetonitrile/H2O 5 pmol/μL in 5% (v/v) acetonitrile/H2O Lyophilized Shipment In solution on wet ice In solution on wet ice Dry at room temperature Concentration precision ±5–10% ±10–25% NA Peptide length* Up to 15 amino acids Purity >97% Isotopic enrichment >99% Standard (light) peptides available Yes Delivery format Glass vial Production time 5–7 weeks standard AQUA Quality control MALDI-TOF MS, HPLC, quantitative AAA Modifications Single or double phosphorylation (pY, pT, or pS) Cysteine carbamidomethylation (CAM) Methionine sulfoxide [Met(O)] Other modifications available on request Optional services Additional light amino acids to extend peptide length Additional heavy amino acids within the peptide sequence Multiple solvents, concentrations, and aliquot sizes available * Please inquire about longer peptide lengths. Learn more at thermofisher.com/heavypeptide Mass spectrometry thermofisher.com/proteinbiology 93 Validated reagents for robust and reproducible sample processing Thermo Scientific™ reagents and kits are optimized for mass spectrometry sample preparation for every step of the process, from depletion strategies to reduce complexity, to protein extraction, enrichment, and cleanup for the isolation of low-abundance proteins. Post-digestion, peptide enrichment, fractionation, and cleanup steps are often critical for proper analysis. The Thermo Scientific™ Pierce™ Quantitative Colorimetric and Fluorometric Peptide Assays enable sample normalization following digestion. Highlights: • Comprehensive—reagents and kits for every step of the sample preparation workflow, both before and after protein digestion • Optimized—reagents and kits are designed to maximize yield without sacrificing quality • Versatile—products available as complete kits or as stand-alone reagents, featuring the Thermo Scientific™ EasyPep™ and High-Select™ lines of sample prep kits • Compatible—kits are designed to be compatible with mass spectrometry analysis • Validated—products have been fully tested, and processed samples have been analyzed using Thermo Scientific™ mass spectrometers Protein sample preparation High-Select Abundant Protein Depletion Reagents and Phosphopeptide Enrichment Kits The High-Select product line offers kits and resins for better detection of low-abundance proteins through sample depletion and enrichment. Thermo Scientific™ High-Select™ HSA/Immunoglobulin and Top14 Abundant Protein Depletion Resins deplete the abundant protein and antibody components of human plasma samples, helping to prepare the samples for mass spectrometry or 1D or 2D electrophoresis (Table 3). Thermo Scientific™ High-Select™ Phosphopeptide Enrichment Kits and Resins available in agarose and magnetic bead formats enable efficient and selective enrichment of phosphorylated peptides for mass spectrometry analysis. Highlights: • Optimized—protocols and reagents designed to maximize results in a fraction of the processing time • High quality—greater than 95% depletion of the top serum/plasma proteins and phosphopeptide recovery with >90% selectivity (Figures 6–8) • Flexible—kits and resins provided in several formats to address sample input and throughput • Scalable—compatible with automation Table 3. Proteins removed by High-Select abundant protein depletion spin columns. HSA/immunoglobulin Top14 columns Albumin Albumin α-Acid glycoprotein IgG IgG Fibrinogen IgA IgA Haptoglobin IgM IgM α1-Antitrypsin IgD IgD α2-Macroglobulin IgE IgE Transferrin IgG (light chains) IgG (light chains) Apolipoprotein A-1 94 Mass spectrometry thermofisher.com/proteinbiology High-Select HSA/Immunoglobulin M FT Serum Serum E FT E High-Select Top 14 3,932 Total unique peptides 70 88300 (8 μg) 9,000 6,000 3,000 0 100 80 60 40 20 0 No. of peptides Selectivity (%) 6,045 99 A32992 (33 μg) Phosphopeptides Selectivity (%) Figure 6. High-Select protein depletion spin columns remove proteins to enable visualization of low-abundance proteins. Human serum (10–20 μL) was loaded onto each resin and processed according to protocols. Total protein in the depleted fractions was estimated using the Thermo Scientific™ Pierce™ BCA Protein Assay Kit. The total amount of albumin in the depleted fractions was determined using the AssayMax™ Human Albumin ELISA Kit. FT = flowthrough (i.e., depleted sample); E = eluate (i.e., proteins that were bound by the resin, plus stripped affinity antibodies of the column). With the top 14 proteins removed, low-abundance proteins are now visible in each depleted sample lane (FT). Figure 7. The High-Select Phosphopeptide Enrichment Kit (Cat. No. A32992) improves selectivity and yield over the Pierce Fe-NTA Phosphopeptide Enrichment Kit (Cat. No. 88300). The average selectivity for phosphopeptides is 99% with a 3- to 4-fold improvement in peptide yield. Protein digests (1 mg) from nocadazole-arrested HeLa cells were used for the enrichment according to instructions. Eluted peptides were analyzed using a Thermo Scientific™ EASY-Spray™ C18 LC Column (3 μm, 150 mm) on a Thermo Scientific™ Orbitrap™ Fusion Tribrid Mass Spectrometer. Figure 8. Phosphopeptide enrichment from nocodazole-treated HeLa cell digests. 1 mg of digest was enriched using 25 μL of Fe-NTA magnetic beads before LC-MS analysis (n = 3). Enrichment using Thermo Scientific™ High-Select™ Fe-NTA Magnetic Agarose beads had higher phosphopeptide identifications and equivalent phosphospecificity (97%) compared to another supplier’s resin. 0 20 40 60 80 100 0 3,000 6,000 9,000 12,000 15,000 High-Select Fe-NTA kit Supplier A Phosphospecificity (%) Phosphopeptide identifications Phosphopeptides Phosphospecificity Mass spectrometry thermofisher.com/proteinbiology 95 Simplified sample preparation solutions for mass spectrometry Sample preparation of peptides for MS analysis is complex, with numerous steps and nonstandard protocols, resulting in variable sample quality and poor reproducibility. To address these issues, the Thermo Scientific™ EasyPep™ MS Sample Prep Kits have been designed using a standardized workflow to improve reproducibility while also saving hands-on and processing time. EasyPep mini, maxi, and 96-well plate MS sample prep kits Traditional workflow 50 steps ~48 hr 24 steps 2–4 hr EasyPep kit workflow Lysis 0.2 1 1 15 15 0.2 Reduction Alkylation Protein cleanup Protein digestion Peptide cleanup Lysis Reduction/ alkylation Protein digestion Peptide cleanup (hr) (hr) 0.1 0.2 1–3 0.2 Figure 9. Comparison of sample preparation procedures for MS analysis, using a traditional workflow and EasyPep MS Sample Prep Kits. Compared to a traditional workflow, the EasyPep MS Sample Prep Kits produce high-quality peptides in less time, and with half the number of manipulation steps. Figure 10. Comparison of the EasyPep and conventional methods for processing TMT-labeled multiplexed samples. The EasyPep kit resulted in more peptide and protein identifications with high TMT labeling efficiency (N terminus and lysine) compared to a conventional acetone precipitation workflow. Highlights: • Complete—includes preformulated reagents, and an optimized protocol for processing up to 20 samples (mini column), 8 samples (maxi column), or one 96-well plate • Compatible—reagents and protocol designed for compatibility with upstream and downstream processes, including TMT and TMTpro labeling • Flexible—reagents and protocol have been verified using cells, plasma, and tissue samples for 10 μg to 2 mg samples • Time-saving—sample processing has been reduced from more than 1 day to less than 4 hours, with improved results (Figures 9 and 10) Protein group identifications Unique peptide identifications TMT labeling e ciency TIC area 19,120 14,848 3,459 2,797 99.7 96.6 5.8 x 1012 5.6 x 1012 Conventional workflow EasyPep kit 25,000 20,000 15,000 10,000 5,000 0 7 x 1012 6 x 1012 5 x 1012 4 x 1012 3 x 1012 2 x 1012 1 x 1012 0 4,000 3,500 6,000 2,500 2,000 1,500 1,000 500 0 120% 100% 80% 60% 40% 20% 0% Conventional workflow EasyPep kit Conventional workflow Conventional EasyPep kit workflow EasyPep kit Protein group identifications Unique peptide identifications TMT labeling e ciency TIC area 19,120 14,848 3,459 2,797 99.7 96.6 5.8 x 1012 5.6 x 1012 Conventional workflow EasyPep kit 25,000 20,000 15,000 10,000 5,000 0 7 x 1012 6 x 1012 5 x 1012 4 x 1012 3 x 1012 2 x 1012 1 x 1012 0 4,000 3,500 6,000 2,500 2,000 1,500 1,000 500 0 120% 100% 80% 60% 40% 20% 0% Conventional workflow EasyPep kit Conventional workflow Conventional EasyPep kit workflow EasyPep kit Protein group identifications TMT labeling e ciency 3,459 2,797 99.7 96.6 4,000 3,500 6,000 2,500 2,000 1,500 1,000 500 0 120% 100% 80% 60% 40% 20% 0% Conventional workflow EasyPep kit Conventional workflow EasyPep kit A B C How-to video for EasyPep Mini MS Sample Prep Kit: Learn more at thermofisher.com/easypep Use this rapid mass spec sample prep kit for protein extraction, digestion, and peptide cleanup in 2–4 hours. 96 Mass spectrometry thermofisher.com/proteinbiology Optimized and ready-to-use controls, standards, and calibration solutions Thermo Scientific™ Pierce™ calibration solutions for mass spectrometry are ready-to-use liquid formulations that can quickly calibrate Thermo Scientific™ LC-MS instrumentation. In addition to calibration solutions, standards for sensitivity assessment, chromatographic performance, determination of digestion efficiency, top-down method development, or as a control for sample analysis are available (Tables 4 and 5). Also available are high-purity, validated mobile phases and acidic ion-pairing agents that are essential for achieving effective and reproducible liquid chromatography (LC) separation of peptides for electrospray ionization (ESI) MS. Instrument calibration Highlights: • Optimized—products developed to maximize LC-MS instrument performance (Figures 11 and 12) • Convenient—calibration solutions, standards, and solvent blends require little or no preparation • High purity—all reagents provided in nonleachable containers • Validated—all products have been manufactured in an ISO 9001–certified facility and fully tested using Thermo Scientific™ mass spectrometers • Long shelf life—products stable for a minimum of 1 year if stored properly Table 4. Choose the right calibration solution for your platform. Thermo Scientific™ calibration solution Components Mass range Calibration mode Storage Recommended Thermo Scientific™ mass spec instrument series Pierce FlexMix Calibration Solution for Auto-Ready Mass Spectrometers 16 components* 50–3,000 Positive and negative Room temperature Orbitrap IQ-X MS Pierce FlexMix Calibration Solution 16 components* 50–3,000 Positive and negative Room temperature Orbitrap Fusion, Orbitrap Fusion Lumos, Orbitrap ID-X MS Pierce LTQ ESI Positive Ion Calibration Solution Caffeine, MRFA, Ultramark 1621 195–1,522 Positive 2–8°C LTQ, LTQ Orbitrap, LXQ, LCQ FLEET, Exactive Pierce LTQ Velos ESI Positive Ion Calibration Solution Caffeine, MRFA, Ultramark 1621, n-butylamine 74–1,522 Positive 2–8°C LTQ Velos/Velos Pro, LTQ Orbitrap Velos/Orbitrap Velos Pro, Orbitrap Elite, Q Exactive, Orbitrap Fusion Tribrid Pierce ESI Negative Ion Calibration Solution SDS, sodium taurocholate, Ultramark 1621 265–1,680 Negative 2–8°C LTQ, LTQ Velos, LTQ Orbitrap, Exactive Pierce Triple Quadrupole Calibration Solution Tyrosine₁ Tyrosine₃ Tyrosine₆ 182–997 Positive 2–8°C TSQ Quantum, TSQ Discovery, TSQ Quantum Access, TSQ Vantage, TSQ Endura, TSQ Quantiva Pierce Triple Quadrupole Calibration Solution, Extended Mass Range 17 components* 69–2,722 (positive) 69–2,934 (negative) Positive and negative 2–8°C TSQ Quantum, TSQ Discovery, TSQ Quantum Access, TSQ Vantage, TSQ Endura, TSQ Quantiva, TSQ Fortis, TSQ Quantis, TSQ Altis * See website for details. Table 5. Choose the right MS standard for your application. Pierce Reserpine Standard Pierce Small Molecule System Suitability Standard Pierce BSA Protein Digest Pierce 6 Protein Digest Pierce Yeast Digest Standard Pierce HeLa Protein Digest Standard Pierce HeLa Digest/PRTC Standard Pierce TMT11plex Yeast Digest Standard Components Reserpine Glycine, atenolol, flumetsulam, atrazine, terfenadine, warfarin, Ultramark 1621, methyl malonic acid, rafoxinide BSA tryptic peptides Lysozyme, BSA, cytochrome C, alcohol dehydrogenase, β-galactosidase, apotransferrin (multi-species) Yeast lysate peptide digest HeLa lysate peptide digest HeLa digest plus PRTC peptides (Cat. No. 88320, 88321) Yeast peptide digest labeled with TMT11plex label reagents Format Liquid Liquid Lyophilized Lyophilized Lyophilized Lyophilized Lyophilized Lyophilized Primary application Instrument QC Metabolomics system suitability standard Protein positive control Chromatography assessment System suitability standard System suitability standard System suitability standard System suitability standard Complexity of standard + +++ ++ +++ +++ ++++ ++++ ++++ Recommended storage 4°C 4°C –20°C –20°C –20°C –20°C –20°C –20°C Mass spectrometry thermofisher.com/proteinbiology 97 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NL: 6.19E7 NL: 2.62E7 20 0 40 60 80 100 Relative abundance 20 0 40 60 80 100 Relative abundance 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) Figure 12. Example chromatograms of standard and calibration solutions. Base peak chromatogram of 200 ng Thermo Scientific™ Pierce™ HeLa Protein Digest Standard separated using a Thermo Scientific™ Acclaim™ PepMap™ 100 3 μm x 75 μm x 15 cm column with a 2–35% gradient (buffer A: 0.1% formic acid in LCMS-grade water, buffer B: 0.1% formic acid in 100% LCM-grade acetonitrile) at 300 nL/min for 120 minutes and detected on a Thermo Scientific™ LTQ Orbitrap XL™ Mass Spectrometer (top). Thermo Scientific™ Pierce™ Peptide Retention Time Calibration Mixture (10 μL at 5 pmol/μL, before dilution to 0.5 pmol/μL) was analyzed on a Thermo Scientific™ Velos Pro™ Mass Spectrometer System using a Thermo Scientific™ PepMap™ C18 column (75 μm x 15 cm) with a 2–30% gradient of buffer B using buffer A (0.1% formic acid) and buffer B (0.1% formic acid, 99.9% acetonitrile) at 0.3 μL/min (bottom). Learn more at thermofisher.com/mscalibration Figure 11. Pierce FlexMix Calibration Solution chromatograms. Shown are sample chromatograms in positive and negative modes. Test your protein research knowledge Question: The protease of choice for mass spec sample preparation is: A. Chymotrypsin B. Lys-C C. Trypsin D. Pepsin Answer: C 98 Mass spectrometry thermofisher.com/proteinbiology Ordering information Product Quantity Cat. No. Protein quantitation reagents SILAC Protein Quantitation Kit (LysC), RPMI 1640 1 kit A33971 SILAC Protein Quantitation Kit (LysC), DMEM 1 kit A33969 SILAC Protein Quantitation Kit (LysC), DMEM:F-12 1 kit A33970 ¹³C₆ L-Arginine-HCl 50 mg 88210 ¹³C₆ L-Lysine-2HCl 50 mg 89988 NeuCode Lysine-080 50 mg A33613 NeuCode Lysine-521 25 mg A36753 NeuCode Lysine-202 25 mg A36754 RPMI 1640 Medium for SILAC 500 mL 88365 DMEM for SILAC 500 mL 88364 TMTzero Label Reagent 5 x 0.8 mg 90067 TMTsixplex Isobaric Mass Tagging Kit 35 reactions 90064 TMTsixplex Isobaric Label Reagent Set 5 x 0.8 mg (per tag) 90066 2 x 5 mg (per tag) 90068 TMT10plex Isobaric Label Reagent Set 1 x 0.8 mg (per tag) 90110 3 x 0.8 mg (per tag) 90111 1 x 5 mg (per tag) 90406 8 x 0.2 mg (per tag) 90309 TMT11-131C Label Reagent 3 x 0.8 mg A37724 1 x 5 mg A34807 TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent 3 x 0.8 mg (per tag) A37725 1 x 5 mg (per tag) A34808 TMTpro Zero Label Reagent 1 x 5 mg A44518 5 x 0.5 mg A44519 TMTpro 16plex Label Reagent Set 1 x 5 mg (per tag) A44520 1 x 0.5 mg (per tag) A44521 6 x 0.5 mg (per tag) A44522 TMTpro 18plex Label Reagent Set 6 x 0.5 mg A52047 1 x 5 mg A52045 TMTpro-134C & TMTpro-135N Label Reagents 6 x 0.5 mg A52048 1 x 5 mg A52046 Super Heavy TMT Label Reagent 2 mg A43073 Super Heavy TMTpro Label Reagent 2 mg A52040 IodoTMTzero Label Reagent 5 x 0.2 mg 90100 IodoTMTsixplex Label Reagent Set 5 x 0.2 mg (per tag) 90102 AminoxyTMTsixplex Label Reagent Set 5 x 0.2 mg (per tag) 90402 Sample preparation reagents Pierce MS-Compatible Magnetic IP Kit (Protein A/G) 40 reactions 90409 Pierce Albumin Depletion Kit 24 reactions 85160 DSSO (disuccinimidyl sulfoxide) 10 x 1 mg A33545 DSBU (disuccinimidyl dibutyric urea) 10 x 1 mg A35459 DSPP (disuccinimidyl phenyl phosphonic acid, PhoX) 50 mg A52286 TBDSPP (tert-butyl disuccinimidyl phenyl phosphonate, tBu-PhoX) 50 mg A52287 High-Select HSA/Immunoglobulin Depletion Mini Spin Columns 24 columns A36366 High-Select Top14 Abundant Protein Depletion Mini Spin Columns 24 columns A36370 Pierce Trypsin Protease, MS Grade 5 x 20 μg 90057 Pierce Trypsin Protease, MS Grade 5 x 100 μg 90058 Mass spectrometry thermofisher.com/proteinbiology 99 Ordering information (cont.) Product Quantity Cat. No. Pierce LysC Protease, MS Grade 100 μg 90307 Pierce Trypsin/LysC Protease Mix, MS-Grade 100 μg A40009 EasyPep Mini MS Sample Prep Kit 20 reactions A40006 EasyPep 96 MS Sample Prep Kit 96 reactions A44533 Pierce Quantitative Colorimetric Peptide Assay 500 assays 23275 Pierce Quantitative Fluorometric Peptide Assay 500 assays 23290 High-Select Fe-NTA Phosphopeptide Enrichment Kit 30 reactions A32992 High-Select TiO₂ Phosphopeptide Enrichment Kit 24 reactions A32993 High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit 20 reactions A52283 Pierce High pH Reversed-Phase Peptide Fractionation Kit 12 reactions 84868 Pierce Kinase Enrichment Kit with ATP Probe 16 reactions 88310 Pierce Detergent Removal Spin Column, 0.5 mL 25 columns 87777 Pierce Peptide Desalting Spin Columns 25 columns 89852 Pierce C18 Spin Columns 25 columns 89870 Pierce C18 Spin Tips 96 tips 84850 Calibration solutions and standards Pierce LTQ ESI Positive Ion Calibration Solution 10 mL 88322 Pierce LTQ Velos ESI Positive Ion Calibration Solution 10 mL 88323 Pierce ESI Negative Ion Calibration Solution 10 mL 88324 Pierce Triple Quadrupole Calibration Solution, Extended Mass Range 10 mL 88340 Pierce Reserpine Standard for LC-MS 5 x 1 mL 88326 Pierce FlexMix Calibration Solution 10 mL A39239 Pierce Peptide Retention Time Calibration Mixture, 5 pmol/μL 200 μL 88321 Pierce BSA Protein Digest Standard, LC-MS Grade 1 nmol 88341 Pierce 6 Protein Digest Standard, Equimolar, LC-MS Grade 100 pmol 88342 Pierce Intact Protein Standard Mix 5 x 76 μg A33527 Pierce HeLa Protein Digest Standard 5 x 20 μg 88329 Pierce HeLa Digest/PRTC Standard 10 μg A47996 Pierce TMT11plex Yeast Digest Standard 20 μg A40938 Pierce Small Molecule System Suitability Standard 0.25 mL A51740 For more information, or to view additional products, go to thermofisher.com/msreagents Custom peptide synthesis services Thermo Scientific™ custom peptide synthesis services are offered for peptides of 6–70 amino acids, with a synthesis scale of 0.1 mg to 1 g, and with purity from crude to 98%. • Standard Peptide Custom Synthesis Service • HeavyPeptide™ AQUA Custom Synthesis Service • PEPotec™ Immuno Custom Peptide Libraries • PEPotec™ SRM Custom Peptide Libraries thermofisher.com/peptides 100 Mass spectrometry thermofisher.com/proteinbiology Protein crosslinking 104 Protein modification 105 Biotinylation 106 Fluorophore labeling 107 Special packaging options 108 Pierce Premium Grade Reagents 109 Ordering information 110 Protein bioconjugation, crosslinking, and modification A number of techniques for studying the structure and interaction of proteins, as well as for manipulating proteins for use in affinity purification or detection procedures, depend on methods for chemically crosslinking, modifying, or labeling proteins and antibodies. The entire set of crosslinking and modification methods for use with proteins is also referred to as bioconjugation. Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 101 Overview Bioconjugation is the process of chemically joining two or more molecules by a covalent bond where at least one molecule is a biomolecule. This technique utilizes a variety of reagents that contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other biomolecules. The availability of several chemical groups in proteins and peptides make them targets for a wide range of applications including biotinylation, fluorescence dye conjugation, immobilization to solid supports, protein structural studies using crosslinkers, and others. Chemical agents may be used to modify amino acid side chains on proteins and peptides in order to alter charges, block or expose reactive binding sites, inactivate functions, or change functional groups to create targets for crosslinking and labeling. Bioconjugation reagents, crosslinkers, and modification reagents can be described by their chemical reactivity, molecular properties, or applications. Chemical reactivity of bioconjugation and crosslinking reagents The most important property of a bioconjugation reagent or crosslinker is its reactive chemical group(s). The reactive group establishes the method and mechanism for chemical modification, labeling, or crosslinking. Labeling reagents have a reactive moiety at one terminus, such as an NHS ester for amine labeling, and a chemical moiety at the other, such as a fluorescent dye or biotin, for downstream applications. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids. The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryl, carbonyl, and carboxyl groups, and carbohydrates. In addition to targeting functional groups found in biomolecules, bioconjugation reagents and crosslinkers can also employ bioorthogonal groups such as azides and alkynes for click chemistry applications or photoreactive groups for nonselective labeling. Applications Bioconjugation and crosslinking reagents have a variety of applications in life science research and assay development. These include fluorescent labeling of proteins and peptides, protein immobilization onto solid supports, protein–protein conjugation, creation of immunotoxins, and crosslinking for protein structure and interaction studies. Protein conjugates are often designed to enable purification and detection in complex biological samples. Commonly, antibodies are the target of bioconjugation with common labels such as biotin or chemically reactive fluorescent dyes. In this process, NHS ester chemistry is the most widely used method for labeling available lysine residues. Key considerations Bioconjugation and crosslinking reagents are selected based on their chemical reactivities and other chemical properties that affect their behavior in different applications. Key considerations include the chemical specificity of the reactive ends, reaction conditions, and whether further modification to the protein or peptide of interest is required to enable bioconjugation. Other important factors that influence the functionality, specificity, and solubility of the bioconjugation reaction include the spacer arm length, cleavability, composition, and structure (Table 1). 102 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology Packaging options Select a package size or grade based on the scale of your reaction or your requirements. These reagents are available from milligram to kilogram quantities. Milligram Milligram to gram Milligram to gram Gram to kilogram Bulk-size packages available Single tubes Catalog product Premium-grade Large-volume or custom packages Molecular properties Second, choose which features or characteristics are important for your application. Functionality Cleavability Structural modifications N O O O O O N O O O S O S N O O O O N O O O N O O HN O O O F F F F 4 DSS—same reactivity on both ends DSP with disulfide linker for cleavage DBCO—copper-free click moiety Spacer arm composition Spacer arm length Spacer arm structure N O O N O O N O O O O N O O O n O O O O HO NH2 BMH with hydrocarbon spacer AMAS 4.4 Å—short spacer between reactive groups CA(PEG)n—adds solubility in aqueous solutions Table 1. Key considerations for selecting the right bioconjugation reagent. Chemical reactivity First, select a reagent with the functional group(s) to bind your biomolecules of interest. NHS ester reaction Maleimide reaction Amine-containing molecule NHS ester compound Amine bond NHS NH2 + + R O O O O N HO O O R N O NH pH > 7 Sulfhydryl-containing molecule Maleimide compound Thioether bond SH + R O N O R O N S O pH > 6.5–7.5 Applications Select the specific reagent depending on the application (e.g., protein detection, immobilization, or interaction studies). Conjugation Immobilization Protein interaction studies O O N O O N O O S Na+OO O N O O O O O N O O O N O S O O O O N O O O O Sulfo-SMCC DSS—couples proteins to surfaces DSSO—MS-cleavable crosslinker Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 103 Our crosslinking reagents are designed for a variety of techniques including protein–protein conjugation, protein immobilization, antibody–drug conjugation, and protein structural studies. We offer a complete portfolio of crosslinkers tailored to a variety of applications with homobifunctional crosslinkers having the same reactive chemistry at both ends and heterobifunctional crosslinkers having different chemistries at each end. Choosing the right crosslinker depends on the chemical specificity of the reactive ends, reaction conditions, and spacer composition. Depending on the application, it may be desirable to adjust the degree of protein labeling (Figure 1). Highlights: • Wide selection—crosslinkers can target a wide variety of functional groups for tailored conjugation on proteins (amines and sulfhydryls) and bioorthogonal reactivity (Table 2) • Diverse composition—choose from multiple spacer arm lengths, hydrophobicity, and cleavability • Multiple formats—bulk, Thermo Scientific™ No-Weigh™ format, and optimized kit packaging available Protein crosslinking 0.5 1.0 5.0 10 8 6 4 2 0 Degree of labeling (DBCO) [lgG] (mg/mL) 5x Eq TFP Ester- PET4-DBCO 10x 20x 40x Figure 1. The level of DBCO labeling of antibodies or other proteins can be controlled by varying the concentration and ratio of protein to TFP Ester-PEG4-DBCO using the Thermo Scientific™ EZ-Link™ DBCO Protein Labeling Kit. Learn more at thermofisher.com/proteincrosslinking Table 2. Overview of crosslinker reactivities and examples. Reactivity class Target functional group Reactive chemical group Examples Amine-reactive NH2 NHS ester NHS, sulfo-NHS Imidoester DMS, DMP Carboxyl- and amine–reactive COOH Carbodiimide EDC, DCC Sulfhydryl-reactive SH Maleimide SMCC, sulfo-SMCC Haloacetyl SIAB, sulfo-SIAB, SIA Piridyl disulfide PDPH Aldehyde-reactive CHO Hydrazide BMPH, EMCH, PMPH Photoreactive Random Diazirine SDA, sulfo-SDA Aryl azide Sulfo-SANPAH Hydroxyl (nonaqueous)-reactive OH Isocyanate PMPI Azide-reactive N3 Phosphine NHS-azide, NHS-PEGn-azide DBCO/alkyne 104 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology Protein analysis and detection techniques often require more than direct conjugation with a bifunctional crosslinker or activated labeling reagent. For example, in many situations, specialized protein modifications are needed to add molecular mass, increase solubility for storage, or create a new functional group that can be targeted in a subsequent reaction step. Our extensive portfolio of modification reagents enables you to modify your protein for your specific experimental need (Table 3). Highlights: • Convenient—ready-to-use reagents and convenient single-use packaging available • Broad portfolio—extensive offering of reagents to increase solubility, block or derivatize functional groups, and reduce proteins • High quality—products manufactured in ISO 9001– certified facilities Protein modification Learn more at thermofisher.com/proteinmodification Table 3. Overview of key protein modification reagents. Reagent Reactive groups Application 2-Mercaptoethanol Thiol Reducing agent Sodium cyanoborohydride Cyanoborohydride Reduce Schiff base to alkylamine linkage CA(PEG)n Amine Carboxylic acid Reversibly block primary amines (CA(PEG)₄); PEGylation of protein or surface, terminating with a carboxylic acid or primary amine Citrionic anhydride NA Reversibly block primary amines CT(PEG)₁₂ Carboxylic acid Thiol PEGylation of gold or metal surfaces, terminating with a carboxylic acid Cysteine HCl Thiol Reducing agent DTT, Cleland’s reagent Thiol Reducing agent Ethylenediamine 2HCl Amine Addition of amines to protein or surface Guanidine HCl NA Protein denaturant Hydroxylamine NA Deprotecting SATA-modified molecules Iodoacetic acid Iodoacetyl S-carboxymethylation of sulfhydryls (reduced cysteines) MA(PEG)n Amine PEGylation of a protein, oxidized carbohydrate, or surface terminating with a methyl group MM(PEG)12, MM(PEG)24 Maleimide Branched PEGylation of protein or surface, terminating with methyl group MS(PEG)n N-hydroxysuccinimide ester PEGylation of a protein or surface, terminating with methyl group NEM Maleimide Irreversible blocking of sulfhydryl groups SATA N-hydroxysuccinimide ester Modification of primary amines with protected sulfhydryl Sodium meta-periodate Periodate Oxidize carbohydrates for reductive amination Sodium NHS-acetate N-hydroxysuccinimide ester Irreversibly block amines TCEP Phosphine Reducing agent Traut’s reagent Iminothiolane Modify primary amines to contain a free sulfhydryl group Urea NA Reducing agent Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 105 The highly specific interaction of avidin with biotin is a valuable tool in designing nonradioactive purification and detection systems. The extensive line of Thermo Scientific™ biotinylation labeling reagents are designed to be conjugated to an antibody, protein, peptide, cell surface proteins, or nucleic acids (Table 4). Multiple types of biotinylation reagents are available with varied uses preferable for specific applications, so we’ve developed easy-to-use guides for selecting the right reagent. In addition to bulk packaging, we offer the easy-to-use No-Weigh format and optimized kits of our biotinylation reagents. Highlights: • Multiple reactivities—wide selection of amine-, sulfhydryl-, carbonyl-, UV-, and click-reactive biotinylation reagents • Reversible and cleavable—desthiobiotin and cleavable biotin for capture and release applications • Application-specific—reagents designed for specific applications such as cell surface protein labeling or sitespecific antibody labeling Biotinylation Learn more at thermofisher.com/biotinlabeling Table 4. Recommended biotinylation reagents for specific targets and applications. Target Example Reactivity Application Antibody Sulfo-NHS-LC-biotin Amine-reactive Biotin-PEG ELISA, blotting, imaging 4-hydrazide Carbohydrate-reactive SiteClick Biotin Antibody Labeling Kit Azide- and alkyne-reactive Purified protein Sulfo-NHS-SS-biotin Amine-reactive ELISA, blotting, or affinity Maleimide-PEG₂-biotin Sulfhydryl-reactive purification of receptor or antigen Biotin-PEG4-hydrazide Carbohydrate-reactive Purified peptide NHS-PEG4-biotin Amine-reactive ELISA, blotting, or affinity Maleimide-PEG purification of receptor or antigen 2-biotin Sulfhydryl-reactive Amine-PEG2-biotin Carbohydrate- and amine-reactive Cell surface proteins Cell Surface Protein Isolation Kit Amine-reactive Affinity purification or removal of cell surface receptor ligands DNA/RNA or oligonucleotides Psoralen-PEG₃-biotin Random Capture or detection of oligonucleotides in ELISA-type Amine-PEG₂-biotin Carbohydrate- and amine-reactive applications or affinity purification 106 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology Fluorophores are invaluable tools for detecting protein location and activation, identifying protein complex formation and conformational changes, and monitoring biological processes in vivo. The vast selection of fluorophores today provides greater flexibility, variation, and fluorophore performance for research applications than ever before. Our large selection of superior Invitrogen™ Alexa Fluor™ dyes across the spectrum enables labeling of a variety of molecules including peptides, proteins, and antibodies. Dyes are compatible with fluorescence microscopy, flow cytometry, high-content analysis, high-density arrays, in vivo imaging, immunoblotting, and immunostaining applications. Highlights: • Superior brightness—conjugates made with Alexa Fluor dyes produce fluorescence output that surpasses that of spectrally similar fluorophore-labeled conjugates • Excellent photostability—lower photobleaching rates allow for more time for image capture • Across the spectrum—dyes span from the near-UV, visible, and near-IR spectrum Fluorophore labeling Learn more at thermofisher.com/fluorescentlabeling Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 107 Optimized kits We’ve packaged many of our popular fluorophores, biotinylation reagents, and crosslinkers in easy-to-use optimized kits for labeling and crosslinking. Each kit contains everything needed to label a protein or antibody with the desired label including the reagents, conjugation buffer, purification system, and an easy-tofollow, step-by-step protocol for conjugation. Highlights: • Scalable—kits are designed for labeling as little as 100 μg of protein and up to 10 mg • Easy—everything included with an optimized workflow for labeling • Wide selection—kits are available for labeling with Alexa Fluor dyes, biotin, and crosslinkers No-Weigh packaging format Bioconjugation reagents are highly reactive molecules and therefore may be sensitive to water, light, oxygen, or other surrounding conditions. They may form unstable intermediates that lead to undesired side products of the intended reactions. Therefore, it is critical to minimize the exposure of bioconjugation reagents to these negative environmental factors to obtain the highest yields and quality possible. To mitigate these risks, we offer specific packaging and quality grades for your bioconjugation reagents. In addition to these options, our bioconjugation reagents are available in multiple pack sizes. Custom packaging and pack sizes are also available by request. With the convenient Thermo Scientific™ No-Weigh™ format, a ready-to-use solution can be made quickly and conveniently. This unique packaging format helps to eliminate the need to weigh out small volumes of dry chemicals. Once reconstituted, the reagent is ready to use at the desired concentration. Avoid weighing hassles and wasting precious reagents with our single-use No-Weigh packaging format. Special packaging options Learn more at thermofisher.com/no-weigh Learn more at thermofisher.com/labelingkits Highlights: • Helps save time—avoid weighing chemicals; just add water, buffer, or solvent to create a working solution in seconds • Helps reduce waste—small working aliquots limit the amount of unused material discarded • Always fresh—working solution is ready to use at desired concentration; no need to store stock solutions 108 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology Pierce Premium Grade reagents Thermo Scientific™ Pierce™ Premium Grade reagents are an ideal choice for applications where product integrity and risk minimization are critical. Compared to standard-grade reagents, Pierce Premium Grade reagents provide clearly defined quality by including batch-specific information such as quality assurance review, lot sample retention, and change control notification (CCN), as well as an enhanced level of analytical testing and product characterization. Highlights: • Quality reagents—high-purity reagents that can be used to create high-quality activated derivatives, labeled proteins, and bioconjugates • Product integrity—enhanced level of testing and characterization • Lot retention—ample supply of past lots retained to ensure future process testing Learn more at thermofisher.com/premium-grade Test your protein research knowledge Question: What kind of bond is involved in the bioconjugation of two molecules? A. Hydrogen B. Covalent C. Cohesive D. Ionic Answer: B Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 109 Ordering information Product Quantity Cat. No. Crosslinkers NHS (N-hydroxysuccinimide) 25 mg 24500 Sulfo-NHS (N-hydroxysulfosuccinimide) 500 mg 24510 Pierce EDC, No-Weigh Format 10 x 1 mg A35391 EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) 25 g 22981 BS3 (bis(sulfosuccinimidyl)suberate) 50 mg 21580 SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) 50 mg 21857 Pierce SMCC, No-Weigh Format 10 x 1 mg A35394 Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) 50 mg 22322 SMPH (succinimidyl 6-((β-maleimidopropionamido) hexanoate)) 50 mg 22363 Pierce DSG, No-Weigh Format 10 x 1 mg A35392 DSG (disuccinimidyl glutarate) 50 mg 20593 Pierce DSP, No-Weigh Format 10 x 1 mg A35393 DTSSP (3,3´-dithiobis(sulfosuccinimidyl propionate)) 50 mg 21578 DMS (dimethyl suberimidate) 1 g 20700 DMP (dimethyl pimelimidate) 1 g 21667 N,N´-dicyclohexylcarbodiimide 100 g 113901000 Sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl)aminobenzoate) 50 mg 22327 SIA (succinimidyl iodoacetate) 50 mg 22349 PDPH (3-(2-pyridyldithio)propionyl hydrazide) 50 mg 22301 BMPH (N-β-maleimidopropionic acid hydrazide) 50 mg 22297 Sulfo-SDA (sulfo-NHS-diazirine) (sulfosuccinimidyl 4,4´-azipentanoate) 50 mg 26173 Sulfo-SANPAH (sulfosuccinimidyl 6-(4´-azido-2´-nitrophenylamino)hexanoate) 50 mg 22589 PMPI (N-(p-maleimidophenyl) isocyanate) 50 mg 28100 NHS-PEG₄-Azide 100 mg 26130 EZ-Link TFP Ester-PEG₄-DBCO 25 mg C20039 View all available products at thermofisher.com/crosslinking Protein modification reagents Pierce 2-Mercaptoethanol 10 x 1 mL 35602BID AminoLink Reductant (sodium cyanoborohydride) 2 x 1 g 44892 CA(PEG)4 100 mg 26120 Citraconic Anhydride 25 g 154790250 CT(PEG)12 100 mg 26133 Pierce Cysteine-HCl 5 g 44889 Pierce DTT, Cleland’s Reagent 5 g 20290 Guanidine-HCl 500 g 24110 Pierce Hydroxylamine-HCl 25 g 26103 Pierce Iodoacetic Acid 500 mg 35603 Pierce BS(PEG)5, No-Weigh Format 10 x 1 mg A35396 Pierce SM(PEG)2, No-Weigh Format 10 x 1 mg A35397 Pierce SM(PEG)12, No-Weigh Format 10 x 1 mg A35398 MM(PEG)12 100 mg 22711 MS(PEG)4 100 mg 22341 MT(PEG)4 100 mg 26132 Pierce NEM (N-ethylmaleimide) 25 g 23030 Pierce SATA (N-succinimidyl S-acetylthioacetate) 50 mg 26102 110 Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology Product Quantity Cat. No. Pierce TCEP-HCl, No-Weigh Format 10 x 1 mg A35349 Bond-Breaker TCEP Solution, Neutral pH 5 mL 77720 Pierce Traut’s Reagent (2-iminothiolane HCl) 500 mg 26101 Urea 1 kg 29700 View all available products at thermofisher.com/proteinmodification Biotinylation reagents EZ-Link NHS-PEG12-Biotin, No-Weigh Format 10 x 1 mg A35389 EZ-Link NHS-PEG12-Biotin 500 mg 21313 EZ-Link Sulfo-NHS-LC-Biotin 100 mg 21335 EZ-Link Sulfo-NHS-LC-LC-Biotin, No-Weigh Format 10 x 1 mg A35358 EZ-Link Sulfo-NHS-LC-LC-Biotin 50 mg 21338 EZ-Link Sulfo-NHS-SS-Biotin 100 mg 21331 EZ-Link NHS-LC-Biotin 50 mg 21336 EZ-Link Maleimide-PEG₂-Biotin 50 mg 21901BID EZ-Link Amine-PEG₃-Biotin 50 mg 21347 EZ-Link NHS-PEG₄-Biotin 50 mg 21362 EZ-Link HPDP-Biotin 50 mg 21341 SiteClick Biotin Antibody Labeling Kit 1 kit S20033 View all available products at thermofisher.com/biotinlabeling Fluorescent reactive dyes Alexa Fluor 350 NHS Ester 5 mg A10168 Alexa Fluor 488 NHS Ester 5 mg A20100 Alexa Fluor 532 NHS Ester 5 mg A20101MP Alexa Fluor 546 NHS Ester 5 mg A20102 Alexa Fluor 555 NHS Ester 5 mg A20109 Alexa Fluor 568 NHS Ester 5 mg A20103 Alexa Fluor 594 NHS Ester 5 mg A20104 Alexa Fluor 633 NHS Ester 5 mg A20105 Alexa Fluor 647 NHS Ester 5 mg A20106 Alexa Fluor 660 NHS Ester 1 mg A20007 Alexa Fluor 700 NHS Ester 5 mg A20110 Alexa Fluor 750 NHS Ester 5 mg A20111 View all available products at thermofisher.com/fluorescentlabeling Protein bioconjugation, crosslinking, and modification thermofisher.com/proteinbiology 111 Cryo-EM sample preparation products 115 Ordering information 118 Cryo-EM sample preparation Obtaining a protein structure is critical to understanding protein function. In recent years cryo-electron microscopy (cryo-EM) has seen rapid adoption among structural biologists due to its ability to image even the most difficult targets, like membrane proteins and complex molecular machinery, as well as the achievable resolution that is getting ever closer to matching that of X-ray crystallography. Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Cloning and protein expression Protein gel electrophoresis Mass spectrometry Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Western blotting 112 Cryo-EM sample preparation thermofisher.com/proteinbiology Cryo-EM techniques Cryo-EM cools samples to cryogenic temperatures so quickly that it prevents water molecules from crystalizing, preserving the native sample structure. Once frozen, a range of EM techniques can be used to visualize the specimen in 3D at a variety of resolutions—including near-atomic resolution—allowing for deeper, more comprehensive insights than previously possible. SPA Single particle analysis (SPA) enables near-atomic structural determination of challenging proteins and protein complexes without the need for crystallization. In SPA, purified proteins or protein complexes are suspended in amorphous (vitreous) ice through rapid plunge freezing, which preserves the native structures of the samples. Transmission electron microscopy (TEM) is then used to collect numerous 2D snapshots of the samples. As the proteins are oriented randomly within the ice, these images show the sample at various angles, and can be recombined into a high-resolution 3D reconstruction of the sample. MicroED Microcrystal electron diffraction (MicroED) enables fast, high-resolution, structural determination of small molecules and proteins. Atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture. Data is acquired by cryo-TEM, using electrons as the incident beam. Since MicroED is a diffraction technique, samples need to be crystallized using the same methods that are found in X-ray crystallography. However, much smaller crystals (~100 nm in size) can be used in MicroED because the interaction of the crystal with electrons is much stronger than its interaction with X-rays. This may significantly shorten the sample preparation process and allow for the analysis of crystals that are too small to diffract with other methods. Cryo-ET Cryo-electron tomography (cryo-ET) provides label-free, fixation-free, nanometer-scale imaging of a cell’s interior in 3D and visualizes protein complexes within their physiological environments. Using a correlative light and electron microscopy approach allows targeting of tagged proteins by fluorescence microscopy before subsequent cryo-EM higher-resolution imaging. Many cells are too thick for electrons, so the vitrified cells must be thinned with a cryo-focused ion beam (cryo-FIB) microscope prior to imaging in a transmission electron microscope. Protein sample preparation for cryo-EM analysis The most commonly used cryo-EM method today is SPA, in which purified proteins are imaged to high-resolution 3D structures. SPA relies on near instantaneous freezing of proteins from solution to a thin layer of noncrystalline ice on a grid in a process called vitrification. This process allows for capturing the protein in its native state and providing a protective environment for subsequent electron-based imaging at high vacuum in the transmission electron microscope. Sample preparation is currently a major bottleneck in SPA. Even though semiautomated vitrification robots like the Thermo Scientific™ Vitrobot™ Mark IV System help to make parts of the grid preparation more robust, the process can still prove challenging as many different parameters contribute to the quality of the prepared grid. When beginning cryo-EM sample preparation, it is critical to have a well-characterized and homogeneous sample, preferably tested by negative stain imaging or mass spectrometry. Nevertheless, even purified proteins that are stable and structurally intact can behave differently in a thin vitreous ice layer, exhibiting unwanted behavior such as denaturation, aggregation, or preferred orientation. Due to the unique properties of each protein, multiple rounds of optimization are often necessary, where vitrification parameters, grid types, or additives are adjusted before the optimal condition for high-resolution data collection are found (i.e., particles are structurally intact, randomly oriented, and equally distributed). Currently, sample optimization is often performed in a nonsystematic way, extending optimization time over multiple days or even weeks. In order to support the successful preparation of cryo-EM samples, we offer solutions for every step of the cryo-EM workflow to help to train new users, optimize sample conditions, and provide standards that can be used for benchmarking or workflow optimization (Figure 1). Cryo-EM sample preparation thermofisher.com/proteinbiology 113 Figure 1. Sample preparation workflows for high-resolution cryo-EM. s Single-particle analysis MicroED Cryo-ET Protein expression • Mammalian cell expression • Cell-free expression Cell growth Protein extraction and isolation • Cell lysis • Detergents for protein solubilization Protein purification • Recombinant protein purification • Biotin affinity purification • NanoDrop spectrophotometers Protein clean-up • Zeba spin desalting columns • Protein concentrators Cryo-EM standards and training • VitroEase Apoferritin Standard • VitroEase Training Kit Crystal growth EM sample optimization • VitroEase Buffer Screening Kit • EM-compatible detergents Grid selection Negative stain screening • VitroEase Methylamine Vanadate • VitroEase Methylamine Tungstate Vitrification • Vitrobot Mark IV System Lamellae preparation • Cryo-FIBs Cryo-EM • Tundra Cryo-TEM • Krios G4 Cryo-TEM • Glacios 2 Cryo-TEM 114 Cryo-EM sample preparation thermofisher.com/proteinbiology Cryo-EM sample preparation products VitroEase Cryo-EM Training Kit and Apoferritin Standard The Thermo Scientific™ VitroEase™ Cryo-EM Training Kit and Apoferritin Standard provide a complete solution for the successful preparation of grids for cryo-EM SPA. Highlights: • Positive control sample—highly pure apoferritin protein verified for use in cryo-EM grid preparation and analysis • High-quality—grids and accessories from trusted sources • Convenient—ready-to-use protein standard with step-by-step instructions for successful grid preparation The VitroEase Cryo-EM Training Kit has been designed to provide the necessary components to get started with cryo-EM analysis of protein samples. The kit includes a positive control protein and commonly used accessories used in grid preparation, including tweezers for manipulation, grids, and grid boxes for storage. Detailed step-by-step instructions guide the user through the lengthy vitrification process. The VitroEase Apoferritin Standard has been designed specifically to meet the stringent needs for high protein purity, minimal protein aggregation, and intact protein structure. Apoferritin, a 24-subunit protein (474 kDa), is considered the gold standard for cryo-EM SPA (Figure 2). The standard is available in the kit and sold separately. Together, the control and components may be used as a comparison for success for new users or for quality control for experienced users. Figure 2. Representative micrographs at (A,C) grid square level and (B,D) hole level of two different VitroEase Apoferritin Standard batches prepared with the recommended vitrification settings. Cryo-EM sample preparation thermofisher.com/proteinbiology 115 VitroEase Buffer Screening Kit The Thermo Scientific™ VitroEase™ Buffer Screening Kit provides preformulated buffers and detergents with a comprehensive screening strategy to identify optimal protein conditions for cryo-EM SPA. Highlights: • Ease of use—preformulated buffers and detergents at 10X concentration • Comprehensive—14 buffers and 6 detergents commonly used for cryo-EM sample preparation provided with an optimized screening strategy • Flexible—range of salts, pH, and detergent formulations for sample optimization of 400 grids • Validated—functionally validated using cryo-EM SPA Cryo-EM sample screening evaluates protein behavior in various buffers and additive conditions with the end goal of identifying the optimal conditions in which the protein is stable, randomly oriented, and equally distributed in ice. Kit buffers are provided with a pH range from 3.6 to 8.9, premixed with various salts and divalent cations. The comprehensive sample screening strategy uses two rounds of screening and 24 grids, taking approximately two days to process (Figure 3). In the first round, optimal protein concentration, buffer, and pH conditions are determined. In the second round, detergents are further optimized. A condensed strategy is also provided using 8 grids and taking one day to process, which can be used when little sample is available or for use with side-entry or semiautomated sample loading systems like the Thermo Scientific™ Tundra™ Cryo-TEM. The recommended screening strategies are designed for use with the Thermo Scientific™ Vitrobot™ Mark IV System and Thermo Scientific™ Krios™, Glacios™, and Talos Arctica™ microscopes with autoloader systems and EPU multigrid. Purified protein Selecting buers and detergents Adding buers/detergents to the protein Cryo-EM grid vitrification Vitribot EPU Multigrid First screening round Second screening round Sample prep Microscopy Biochemistry optimization Figure 3. Comprehensive cryo-EM sample screening strategy. 116 Cryo-EM sample preparation thermofisher.com/proteinbiology VitroEase negative stains Thermo Scientific™ VitroEase™ Methylamine Tungstate and Thermo Scientific™ VitroEase™ Methylamine Vanadate Negative Stains are easy, ready-to-use negative stains for electron microscopy (EM) analysis. Highlights: • Provided as a stable, ready-to-use 2% stain solution • Easy to use with straightforward staining protocol • Uniform, electron-dense stain for EM • Dries without crystallization, ideal for EM grids VitroEase Methylamine Tungstate Negative Stain is a ready-to-use stain with excellent spreading qualities with high density for good contrast. It wets both grid films and specimens well at neutral pH. This stain does not damage delicate structures as much as phosphotungstate (PTA) and has been found to be useful for negative staining of macromolecules, viruses, and membranes. VitroEase Methylamine Vanadate Negative Stain is a ready-to-use stain with excellent spreading properties at a near-neutral pH of 8. It produces a light, uniform negative stain for high-resolution fine structure visualization and does not denature protein samples. This stain is also stable and nonvolatile in the electron beam, making it more resistant than uranyl acetate negative stain. Figure 4. TEM images of negative-stained apoferritin using the VitroEase Methylamine Tungstate and VitroEase Methylamine Vanadate Negative Stains. Cryo-EM sample preparation thermofisher.com/proteinbiology 117 Vitrobot Mark IV System The Thermo Scientific™ Vitrobot™ Mark IV System assists in robustly preparing grids for Cryo-EM. The instrument is fast and easy to use and allows the preparation of cryo-EM grids for various cryo-EM applications and sample types, such as purified proteins for SPA, cells for cryo-ET, and crystals for MicroED. Sample preparation is an important step in the cryo-EM workflow, and preparing good quality grids for subsequent imaging can be a daunting task. The Vitrobot Mark IV System helps the user by automating the vitrification process, while providing precise but flexible control over critical parameters. By making use of a temperature- and humidity-controlled climate chamber, the system provides a controlled environment for vitrification that prevents cooling and concentration artifacts that can be introduced with manual or “open space” vitrification systems. Learn more at thermofisher.com/em-sample-prep Highlights: • Fully automated, reproducible vitrification of aqueous suspensions • Precise control of critical process parameters • Enclosed environmental chamber • High sample throughput • Easy and flexible user interface • Semiautomated grid transfer Ordering information Product Quantity Cat. No. Sample prep reagents VitroEase Cryo-EM Training Kit 25 preparations A51363 VitroEase Apoferritin Standard 5 x 20 mL A51362 VitroEase Methylamine Tungstate Negative Stain 5 mL A51036 VitroEase Methylamine Vanadate Negative Stain 5 mL A51037 VitroEase Buffer Screening Kit 1 kit A49856 Instrumentation Vitrobot Mark IV System 1 system VITROBOT For information on cryo-EM instruments, visit thermofisher.com/emlifesciences Test your protein research knowledge Question: Which of the following are cryo-EM techniques? (Select all that apply.) A. SILAC B. SRM C. SPA D. SEM Answer: C 118 Cryo-EM sample preparation thermofisher.com/proteinbiology Protein biology 120 Cloning and protein expression 120 Protein sample preparation 121 Protein purification 122 Protein gel electrophoresis 122 Western blotting 123 Immunoassays 123 Mass spectrometry 124 Protein modification and crosslinking 124 Cryo-EM sample preparation 125 Resources You have come to expect high-quality products for your protein research from us, but our support does not stop there. We also provide technical tips, selection guides, and other helpful tools to make your protein research experiments go more smoothly. Resources thermofisher.com/proteinbiology 119 Protein biology Protein Analysis Learning Center The Protein Analysis Learning Center is here to assist you in your quest to understand the proteome, whether you want to review the basics, gain more in-depth knowledge, or discover how to use the latest research tools. Find resources for the different protein analysis techniques starting from protein expression to quantitative mass spectrometry including webinars, articles, protocols, and troubleshooting tips. Access the learning center at thermofisher.com/proteinlearning Protein Degradation Resource Center The Protein Degradation Resource Center includes the latest information and research tools to assist you in evaluating protein degradation by providing product offerings and solutions to your everyday protein degradation questions. Access the resource center at thermofisher.com/proteindegradation Antibodies Learning Center Selecting individual antibody products from among hundreds of options can be a challenge. The Antibodies Learning Center contains educational material designed to empower researchers and technicians with the background knowledge about antibody technologies necessary to develop, select, and use antibodies to advance their research. Access the learning center at thermofisher.com/antibodies-learning-center Cloning and protein expression Protein expression handbook Download this handbook to help you choose the right protein expression system and purification technologies for your specific application and needs. Get tips and tricks when starting an experiment, and find answers to everyday problems related to protein expression. Request your copy at thermofisher.com/expressionguide Protein expression virtual short courses Join our instructors who will discuss the concept, techniques, and optimization strategies for the rapid generation of milligram-to-gram quantities of secreted or intracellular recombinant proteins for therapeutic, functional, and structural studies. The set of courses combines instruction and case studies in an interactive environment. Register at thermofisher.com/gibcoproteinexpressionvirtualshortcourses 120 Resources thermofisher.com/proteinbiology Protein Expression Basics online learning hub Gibco™ Protein Expression Basics is an online hub that brings together educational content and tools to enhance your research. Here you can access our protein expression selection guide, handbook, educational videos, and technical resources. Start here if you are new to protein expression or need to refresh your knowledge. Access the learning hub at thermofisher.com/gibcoproteinexpressionbasics Protein sample preparation Protein assay technical handbook Download this handbook to help select the appropriate protein assay method based on the assay time, sensitivity, compatibility, standard curve linearity, and protein-toprotein variation. Learn about our wide range of colorimetric (copper- or dye-based) and fluorescence protein assays, as well as our more specialized assays to quantify peptides, antibodies, protein modifications, or functional (enzymatic) classes of proteins. Discover tools and strategies to help optimize your protein quantitation assays to help ensure more accurate downstream results. Download the free handbook at thermofisher.com/protein-assay-handbook Protein clean-up technical handbook Learn how to effectively remove contaminants, perform buffer exchange, or concentrate protein samples from 2 μL to 250 mL using various Thermo Scientific™ protein biology tools in this 48-page handbook. Dialyze protein samples securely using Slide-A-Lyzer cassettes and devices. Rapidly desalt samples with high protein recovery using Zeba desalting spin columns and plates. Efficiently extract specific contaminants using resins optimized for detergent or endotoxin removal. Concentrate dilute protein samples quickly using Pierce Protein Concentrators. Download the free handbook at thermofisher.com/proteincleanuphandbook Protein Sample Preparation eLearning Course This free course comprises two interactive modules that were developed to provide a succinct, contextual overview of protein sample isolation using total protein extraction and subcellular fractionation. The module covers methods for purifying whole cell lysates and describes strategies for protein cleanup procedures. Access the free course at thermofisher.com/elearningproteinsampleprep Protein assay technical handbook Tools and reagents for improved quantitation of total or specific proteins Resources thermofisher.com/proteinbiology 121 Protein purification Tools and reagents for recombinant protein purification Download this handbook to learn more about our wide portfolio of immobilized ligands for the purification of 6xHis-, GST-, c-Myc-, HA-, and DYKDDDDK (FLAG™)-tagged proteins. Our broad offering of magnetic beads, magnetic agarose, agarose, Superflow resins, and UltraLink™ resins enable purification and pull-down applications from low-microgram (high-throughput) to low-kilogram (pilot) scales. Our helpful selection guides and relevant data will help you select the best support to meet your needs. Download the free handbook at thermofisher.com/tag-purification-handbook Protein preparation handbook Discover tools for protein extraction, protein cleanup, protein purification, and immunoprecipitation that can help take your protein research to the next level. Download the free handbook at thermofisher.com/proteinprephandbook Protein gel electrophoresis Protein gel electrophoresis handbook This technical handbook lets you discover even more about protein gels, sample preparation, buffers and reagents, standards, electrophoresis chambers, power supplies, and staining. Learn more at thermofisher.com/pagehandbook Protein gel selection guide Use our interactive product selector to find the right gel for your experiment. Access the guide at thermofisher.com/proteingelguide Protein gel conversion tool Easily find Invitrogen™ gels to replace your current precast gels from another supplier. Access the tool at thermofisher.com/proteingelconversion Protein Standards and Ladders Selection Guide Find the right protein ladder for your application and gel system. Use our interactive tool to see the migration of recommended protein ladders in different gel chemistries. Learn more at thermofisher.com/proteinladdersguide 122 Resources thermofisher.com/proteinbiology Western blotting Western blotting handbook This comprehensive, easy-to-use guide covers every procedural western blotting step, from gel transfer to hands-free blot processing systems, imaging and data analysis, multiplexing, western blot quantitation, and more. Learn more at thermofisher.com/westernhandbook Invitrogen™ BlotBuilder™ western blot product selection tool Let us help you select the right tools specifically for your protein and experimental needs. Simply answer a few questions about your protein of interest and review a set of recommended products with a personalized western blot protocol. Access the tool at thermofisher.com/blotbuilder Protein Electrophoresis and Western Blotting Education Center Gaining publication-quality results immediately is not exactly the norm when performing western blotting. Access resources to learn about protein gel electrophoresis and western blotting methods, from webinars to quick tips and tricks. Whether you are new to western blotting or an experienced researcher looking to expand your knowledge, consider this center to help you get better western results and succeed sooner. Access resources at thermofisher.com/westerneducation Immunoassays ELISA troubleshooting guide Is your ELISA not working? Invitrogen™ ELISA kits are built to be easy, but things can still go wrong. To help, we’ve put together a collection of troubleshooting resources with tips and tricks for the most common challenges. Access the guide at thermofisher.com/elisatroubleshooting ELISA protocols General protocols are available for a standard sandwich ELISA using a 96-well plate for colorimetric (chromogenic) or chemiluminescent detection. View the protocols at thermofisher.com/generalelisaprotocol ELISA selection guide With thousands of ELISA kits to choose from, we’re confident you’ll find the right kit for you. Find your ELISA at thermofisher.com/elisa Resources thermofisher.com/proteinbiology 123 ELISA validation and quality control Validation and quality control are critical aspects for creating reliable ELISA kits. That’s why we put our kits through rigorous testing to ensure they work for you time and time again. Learn more about ELISA validation and QC testing at thermofisher.com/elisavalidation Biomarker quantitation assay guide This guide describes all the options for single- and multiple-biomarker quantitation assays for both protein and mRNA. Learn more at thermofisher.com/biomarkerguide Luminex how-to videos Watch how-to videos to learn tips and techniques for running Luminex® assays. Learn more at thermofisher.com/immunoassayvideos Mass spectrometry Protein sample preparation and quantitation for mass spectrometry This 168-page handbook provides an integrated overview of protein sample preparation, quantitation, and instrument calibration for optimized proteomics workflows using mass spectrometry. Guidelines for selecting the right chromatography products, mass spectrometers, and software for discovery and targeted proteomics applications are provided. Download at thermofisher.com/mshandbook Mass spec analysis technical resources Find a collection of valuable tools to help improve your mass spectrometry results, including handbooks, application-specific white papers, technical notes, late-breaking posters, and helpful webinars. Access now at thermofisher.com/msresources Protein modification and crosslinking Bioconjugation and crosslinking technical handbook This handbook discusses reagents for bioconjugation, crosslinking, biotinylation, and modification of proteins and peptides. Download at thermofisher.com/bioconjugationhandbook 124 Resources thermofisher.com/proteinbiology Biotinylation Reagent Selection Tool This tool provides quick access to customized lists of Thermo Scientific™ biotinylation and desthiobiotin reagents that meet specific criteria, including target functional group, spacer arm length, and solubility. Use the simple dropdown boxes to easily select the optimal biotinylation reagent for your application. Learn more at thermofisher.com/biotinylation-tool Crosslinker Selection Tool This tool provides quick access to customized lists of Thermo Scientific™ Pierce™ crosslinkers that meet specific criteria, including target functional group, solubility, and the ability to permeate the cell membrane. Use the simple dropdown boxes to easily select the optimal crosslinking reagent for your application. Learn more at thermofisher.com/crosslinking-tool Antibody Labeling Kits Selection Guide This guide provides quick access to customized lists of antibody labeling kits that meet specific criteria, including type of label, chemical reactivity, and labeling scale. Use this interactive selection guide to find the right antibody or protein labeling kit for your experiment. Learn more at thermofisher.com/ABlabelingguide Cryo-EM sample preparation eBook: Getting started with cryo-EM Learn more about adopting cryo-EM and how it can advance your research. This eBook was created especially for structural biologists and biochemists who do biomedical research and are interested in obtaining deeper structural and functional insights of macromolecules. Download the eBook at thermofisher.com/cryo-em-ebook eBook: Structure and function with cryo-tomography Cryo-electron tomography allows researchers to access the inner-workings of cells in 3D and at unprecedented nanoscale resolution. Download our eBook to explore a curated collection of scientific publications highlighting the use of cryo-electron tomography for applications in cell biology, neuroscience, microbiology, and virology. Download the book at thermofisher.com/tomographyebook Cryo-EM Learning Center The Cryo-EM Learning Center provides the latest information on cryo-electron microscopy (cryo-EM). This resource hub contains rich and reliable technical content designed for new and experienced researchers who are exploring the capabilities of cryo-EM. Access the learning center at thermofisher.com/cryo-em Resources thermofisher.com/proteinbiology 125 For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AssayMax is a trademark of Assaypro LLC. Bio-Rad, Bio-Spin, ReadyPrep, and TGX are trademarks of Bio-Rad Laboratories. Brij and Tween are trademarks of Croda Americas. MiniTrap, Sepharose, and SpinTrap are trademarks of Cytiva. FLAG, ProteoExtract, and PureProteome are trademarks of Merck KGaA. Gibson Assembly is a registered trademark of SGI-DNA. Luminex, xMAP, MAGPIX, MagPlex, and FLEXMAP 3D are registered trademarks of Luminex Corp. TMT10plex, TMT11plex, TMTsixplex, TMTzero, and TMTpro are trademarks of Proteome Sciences. Triton is a trademark of Union Carbide Corp. COL35719 0623 Learn more at thermofisher.com/proteinbiology