Optimize Your Bioconjugation Strategies

Bioconjugation and crosslinking technical handbook Reagents for bioconjugation, crosslinking, biotinylation, and modification of proteins and peptides Protein labeling and analysis Introduction 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 molecules. 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, metabolic labeling, and protein structural studies. Bioconjugation reagents, crosslinkers, and modification reagents can be described by their chemical reactivity, molecular properties, or by their applications (Table 1). 2 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 H N 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 N R O N H 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 4 Chemical reactivity of crosslinking reagents Introduction 5 Amine-reactive chemical groups 6 Carboxylic acid–reactive 7 chemical groups Sulfhydryl-reactive chemical groups 8 Carbonyl-reactive chemical groups 10 Photoreactive crosslinkers 12 Chemoselective ligation 13 Molecular properties of crosslinking reagents Introduction 14 Homobifunctional and 15 heterobifunctional crosslinkers General reaction conditions 16 Modifications 16 Spacer arm length 18 Spacer arm composition 18 Spacer arm cleavability 18 Spacer arm structure and solubility 19 Applications using bioconjugation and crosslinking reagents Introduction 20 Protein and peptide biotinylation 21 Antibody labeling and crosslinking 22 Synthesis of antibody–drug conjugates 23 Protein immobilization onto 24 solid supports Surface modification using 25 PEG-based reagents Hapten–carrier conjugation for 26 antibody production Protein–protein conjugation 27 Synthesis of immunotoxins 28 Label transfer 28 Subunit crosslinking and protein 28 structural studies Protein interaction and crosslinking 30 using mass spectrometry MS-cleavable crosslinkers 31 (DSSO and DSBU) Enrichable MS crosslinkers 32 PhoX (DSPP) and TBDSPP In vivo crosslinking 34 Metabolic labeling 35 Cell surface crosslinking and biotinylation 36 Cell membrane structural studies 36 Special packaging to meet specific bioconjugation needs Introduction 37 No-Weigh packaging format for 38 bioconjugation reagents Premium-grade protein 39 bioconjugation reagents Bioconjugation resources 40 Glossary of crosslinking terms 42 Ordering information 44 Related handbooks and resources 63 Contents 5 Chemical reactivity of crosslinking reagents Introduction The most important property of a bioconjugation reagent or crosslinker is its reactive chemical group. The reactive group establishes the mechanism for 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 terminus, such as a fluorescent dye or biotin. Crosslinkers contain at least two reactive groups that target functional groups found in biomolecules. The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryls, carbonyls, biorthogonal azides, and alkynes (Figure 1 and Table 2). 5 6 Amine-reactive chemical groups Primary amines (–NH2 ) exist at the N terminus of each polypeptide chain (called the α-amine) and in the side chain of lysine (Lys, K) residues (called the ε-amine). Because of their positive charge at physiological conditions, primary amines are usually outward facing (i.e., on the outer surface of proteins), making them more accessible for conjugation without denaturing protein structure. A number of reactive chemical groups target primary amines (Figure 2), but the most commonly used groups are N-hydroxysuccinimide esters (NHS esters) and imidoesters. N-hydroxysuccinimide esters (NHS esters) NHS esters are reactive groups formed by EDC activation of carboxylate molecules. NHS ester–activated crosslinkers and labeling compounds react with primary amines in slightly alkaline conditions to yield stable amide bonds (Figure 3). The reaction releases N-hydroxysuccinimide, which can be removed easily by dialysis or desalting. Learn more at thermofisher.com/proteincrosslinking H2 N OH O NH2 H2 N OH O HS Lysine Cysteine H N H C O H3 N R C O H N C O HO O H N H C O H N H C O H N C O HO O OH O H N O O SH Gly Glu Gly Asp Gly Cys H2 N R OH O Generic amino acid R Table 2. Popular crosslinker reactive groups for bioconjugation. Reactivity class Target functional group Reactive chemical group Amine-reactive –NH2 • NHS ester • Imidoester • Pentafluorophenyl ester • Hydroxymethyl phosphine Carboxyl-to-amine reactive –COOH Carbodiimide (e.g., EDC) Sulfhydryl-reactive –SH • Maleimide • Haloacetyl (bromo-, chloro-, or iodo-) • Pyridyl disulfide • Thiosulfonate • Vinyl sulfone Aldehyde-reactive (e.g., oxidized sugars, carbonyls) –CHO • Hydrazide • Alkoxyamine • NHS ester Photoreactive (i.e., nonselective, random insertion) Random • Diazirine • Aryl azide Hydroxyl (nonaqueous)-reactive –OH Isocyanate Azide-reactive –N3 • Alkyne • Phosphine Figure 1. Common amino acid functional groups targeted for bioconjugation. O H R Aldehyde R O Epoxide F R Fluorobenzene R S Cl O O Sulfonyl chloride Imidoester CH3 O NH2 R O O O R Anhydride R N C S Isothiocyanate Isocyanate Acyl azide NHS ester R N O N N R N C O R O O N O O R´ O O R O Carbonate Carbodiimide Fluorophenyl ester F F F O F R F O N N C NH Cl− + Figure 2. Reactive chemical groups that target primary amines. NHS ester O O N O O R N HO O O NHS ester NHS reagent Stable conjugate (amide bond) Primary amine on protein O O N O O R N H O R P NH2 + P + pH 7–9 Figure 3. NHS ester reaction scheme for chemical conjugation to a primary amine. R represents a labeling reagent or one end of a crosslinker having the NHS ester reactive group. P represents a protein or other molecule that contains the target functional group (i.e., primary amine). 7 NHS-reactive chemistry NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate–bicarbonate, HEPES, or borate buffers at pH 7.2–8.5 for 30 minutes to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) are not compatible because they compete for the reaction. However, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to stop the reaction. Hydrolysis of the NHS ester competes with the primary amine reaction. The rate of hydrolysis increases with buffer pH and contributes to less-efficient crosslinking in less-concentrated protein solutions. The half-life of hydrolysis for NHS ester compounds is 4–5 hours at pH 7.0 and 0°C. This half-life decreases to 10 minutes at pH 8.6 and 4°C. The extent of NHS ester hydrolysis in aqueous solutions free of primary amines can be measured at 260–280 nm because the NHS by-product absorbs in that range. Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increase the water solubility of crosslinkers containing them. In addition, the charged group prevents sulfoNHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods. Imidoesters Imidoester crosslinkers react with primary amines to form amidine bonds (Figure 4). To ensure specificity for primary amines, imidoester reactions are best done in amine-free, alkaline conditions (pH 10), such as with borate buffer. Because the resulting amidine bond is protonated, the crosslink has a positive charge at physiological pH, much like the primary amine that it replaced. For this reason, imidoester crosslinkers have been used to study protein structure and molecular associations in membranes and to immobilize proteins onto solid-phase supports while preserving the isoelectric point (pI) of the native protein. Although imidoesters are still used in certain procedures, the amidine bonds formed are reversible at high pH. Therefore, the more stable and efficient NHS ester crosslinkers have steadily replaced them in most applications. Imidoester reaction chemistry Imidoester crosslinkers react rapidly with amines at alkaline pH to form amidine bonds but have short half-lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases, making crosslinking more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8 and 10 (Figure 4). Studies using monofunctional alkyl imidates reveal that at pH <10, conjugation can form with just one imidoester functional group. An intermediate N-alkyl imidate forms at the lower pH range and will either crosslink to another amine in the immediate vicinity, resulting in N,N´-amidine derivatives, or it will convert to an amidine bond. At higher pH, the amidine is formed directly without formation of an intermediate or side product. Extraneous crosslinking that occurs below pH 10 sometimes interferes with interpretation of results when thiol-cleavable diimidoesters are used. Carboxylic acid–reactive chemical groups Carboxylic acids (–COOH) exist at the C terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxyls are usually on the surface of protein structure. Carboxylic acids are reactive towards carbodiimides. Carbodiimides (EDC and DCC) EDC and other carbodiimides are zerolength crosslinkers. They cause direct conjugation of carboxylates (–COOH) to primary amines (–NH2 ) without becoming part of the final amide-bond crosslink between target molecules. Imidoester O NH2 R + Imidoester reagent Conjugate (amidine bond) Primary amine on protein NH2 + P + CH3 OH pH 8–9 O NH2 R + NH2 R N H P + Figure 4. Imidoester reaction scheme for chemical conjugation to a primary amine. R represents a labeling reagent or one end of a crosslinker having the imidoester reactive group. P represents a protein or other molecule that contains the target functional group (i.e., primary amine, –NH2 ). Carbodiimide (EDC) N N C NH Cl− + 8 Because peptides and proteins contain multiple carboxyls and amines, direct EDC-mediated crosslinking usually causes random polymerization of polypeptides. Nevertheless, this reaction chemistry is used widely in immobilization procedures (e.g., attaching proteins to a carboxylated surface) and in immunogen preparation (e.g., attaching a small peptide to a large carrier protein). EDC reaction chemistry EDC reacts with carboxylic acid groups to form an active O-acylisourea intermediate that is easily displaced by nucleophilic attack from primary amino groups in the reaction mixture (Figure 5). The primary amine forms an amide bond with the original carboxyl group, and an EDC by-product is released as a soluble urea derivative. The O-acylisourea intermediate is unstable in aqueous solutions. Failure to react with an amine results in hydrolysis of the intermediate, regeneration of the carboxyls, and the release of an N-unsubstituted urea. EDC crosslinking is most efficient in acidic (pH 4.5) conditions and must be performed in buffers devoid of extraneous carboxyls and amines. MES buffer (4-morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate buffers and neutral pH (up to 7.2) conditions are compatible with the reaction chemistry, but with lower efficiency. Increasing the amount of EDC in a reaction solution can compensate for the reduced efficiency. N-hydroxysuccinimide (NHS) or its water-soluble analog (sulfo-NHS) is often included in EDC coupling protocols to improve efficiency or create dry-stable (amine-reactive) intermediates (Figure 6). EDC couples NHS to carboxyls, forming an NHS ester that is considerably more stable than the O-acylisourea intermediate while allowing efficient conjugation to primary amines at physiological pH. EDC is also capable of activating phosphate groups in the presence of imidazole for conjugation to primary amines. The method is sometimes used to modify, label, crosslink, or immobilize oligonucleotides through their 5´ phosphate groups. DCC reaction chemistry and applications DCC (dicyclohexyl carbodiimide) crosslinks carboxylic acids to primary amines in the same manner as EDC. However, because DCC is not water-soluble, it is primarily used in manufacturing and organic synthesis applications rather than in the typical protein research biology laboratory. For example, most commercially available, ready-to-use NHS ester crosslinkers and labeling reagents are manufactured using DCC. Because water is excluded, the resulting NHS ester can be prepared and stabilized as a dried powder without appreciable hydrolysis. DCC is also commonly used in commercial peptide synthesis operations. Sulfhydryl-reactive chemical groups Sulfhydryls (–SH) exist in the side chain of cysteine (Cys, C). Often as part of a protein’s secondary or tertiary structure, cysteines are joined between their side chains via disulfide bonds (–S–S–). These must be reduced to sulfhydryls to make them available for crosslinking by most types of reactive groups. Sulfhydryls are reactive towards maleimides, haloacetyls, and pyridyl disulfides. Carboxylic acid EDC O-acylisourea active ester Crosslinked protein + N N C H N+ Cl− + Primary amine Isourea by-product O NH NH H N+ OH O 1 H2 N 2 N H O 2 1 O O NH N H N+ 1 Figure 5. Carboxyl-to-amine crosslinking using the popular carbodiimide EDC. Molecules 1 and 2 can be peptides, proteins, or any chemicals that have respective carboxylate and primary amine groups. When they are peptides or proteins, these molecules are tens to thousands of times larger than the crosslinker and conjugation arms diagrammed in the reaction. Carboxylic acid EDC crosslinker O-acylisourea intermediate (unstable) Stable conjugate (amide bond) N N C +NH Cl– N HO O O S O– O O Sulfo-NHS Amine-reactive sulfo-NHS ester (dry-stable) H2 O Primary amine Primary amine NH2 OH O 1 O O NH N H N+ Cl– 1 Hydrolysis OH O 1 O O N O O S O– O O 1 NH2 2 N H O 1 2 2 Figure 6. Carboxyl-to-amine crosslinking using the carbodiimide EDC and sulfo-NHS. Addition of NHS or sulfo-NHS to EDC reactions (bottom-most pathway) increases efficiency and enables molecule 1 to be activated for storage and later use. 9 Maleimides Maleimide-activated crosslinkers and labeling reagents react specifically with sulfhydryl groups (–SH) at near-neutral conditions (pH 6.5–7.5) to form stable thioether linkages. Disulfide bonds in protein structures must be reduced to free thiols (sulfhydryls) to react with maleimide reagents. Extraneous thiols (e.g., from most reducing agents) must be excluded from maleimide reaction buffers because they will compete for coupling sites. Short homobifunctional maleimide crosslinkers enable disulfide bridges in protein structures to be converted to permanent, irreducible linkages between cysteines. More commonly, the maleimide chemistry is used in combination with amine-reactive NHS ester chemistry in the form of heterobifunctional crosslinkers that enable controlled two-step conjugation of purified peptides and/or proteins. Maleimide reaction chemistry The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between pH 6.5 and 7.5, resulting in the formation of a stable thioether linkage that is not reversible (Figure 7). In more alkaline conditions (pH >8.5), the reaction favors primary amines and also increases the rate of hydrolysis of the maleimide group to a nonreactive maleamic acid. Maleimides do not react with tyrosines, histidines, or methionines. Thiol-containing compounds, such as dithiothreitol (DTT) and β-mercaptoethanol (BME (also known as 2-mercaptoethanol)), must be excluded from reaction buffers used with maleimides because they will compete for coupling sites. For example, if DTT were used to reduce disulfides in a protein to make sulfhydryl groups available for conjugation, the DTT would have to be thoroughly removed using a desalting column before initiating the maleimide reaction. Interestingly, the disulfide-reducing agent TCEP does not contain thiols and does have to be removed before reactions using maleimide reagents. Excess maleimides can be quenched at the end of a reaction by adding free thiols. EDTA can be included in the coupling buffer to chelate stray divalent metals that otherwise promote oxidation of sulfhydryls (nonreactive). Haloacetyls Most haloacetyl crosslinkers contain an iodoacetyl or a bromoacetyl group. Haloacetyls react with sulfhydryl groups at physiological to alkaline conditions (pH 7.2–9), resulting in stable thioether linkages. To limit free iodine generation, which has the potential to react with tyrosine, histidine, and tryptophan residues, it is best to perform iodoacetyl reactions in the dark. Haloacetyl reaction chemistry Haloacetyls react with sulfhydryl groups at physiological pH. The reaction of the iodoacetyl group proceeds by nucleophilic substitution of iodine with a sulfur atom from a sulfhydryl group, resulting in a stable thioether linkage (Figure 8). Using a slight excess of the iodoacetyl group over the number of sulfhydryl groups at pH 8.3 ensures sulfhydryl selectivity. In the absence of free sulfhydryls, or if a large excess of iodoacetyl group is used, the iodoacetyl group can react with other amino acids. Imidazoles can react with iodoacetyl groups at pH 6.9–7.0, but the incubation must proceed for longer than one week. Histidyl side chains and amino groups react in the unprotonated form with iodoacetyl groups above pH 5 and pH 7, respectively. To limit free iodine generation, which has the potential to react with tyrosine, histidine, and tryptophan residues, iodoacetyl reactions and preparations should be performed in the dark. Iodoacetyl compounds should not be exposed to reducing agents. Maleimide R N O O Maleimide reagent Stable conjugate (thioether bond) Sulfhydryl on protein R N O O R N O O S P SH + P pH 6.5–7.5 Figure 7. Maleimide reaction scheme for chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the maleimide reactive group. P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH). Iodoacetyl group R N I H O Iodoacetyl reagent Conjugate (thioether bond) Sulfhydryl on protein SH R + P + H N O I HI pH >7.5 R H N O S P Figure 8. Iodoacetyl reaction scheme for chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the iodoacetyl or bromoacetyl reactive group. P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH). 10 Pyridyl disulfides Pyridyl disulfides react with sulfhydryl groups over a broad pH range to form disulfide bonds. As such, conjugates prepared using these crosslinkers are cleavable with typical disulfide-reducing agents such as dithiothreitol (DTT). Pyridyl disulfide reaction chemistry Pyridyl disulfides react with sulfhydryl groups over a broad pH range (the optimum is pH 4–5) to form disulfide bonds. During the reaction, a disulfide exchange occurs between the molecule’s –SH group and the reagent’s 2-pyridyldithiol group. As a result, pyridine-2-thione is released and can be measured spectrophotometrically (Amax = 343 nm) to monitor the progress of the reaction. These reagents can be used as crosslinkers and to introduce sulfhydryl groups into proteins. The disulfide exchange can be performed at physiological pH, although the reaction rate is slower than in acidic conditions (Figure 9). Carbonyl-reactive chemical groups Carbonyl (–CHO) groups can be created in glycoproteins by oxidizing the polysaccharide posttranslational modifications with sodium meta-periodate. Hydrazide and alkoxyamine reactive groups target aldehydes. These aldehydes also react with primary amines to form Schiff bases that can be further reduced to form a covalent bond (reductive amination). Carbohydrate modification is particularly useful for creating target sites for conjugation on polyclonal antibodies because the polysaccharides are located in the Fc region. This results in labeling or crosslinking sites located away from antigen binding sites, ensuring that antibody function will not be adversely affected by the conjugation procedure. Carbonyls (aldehydes) as crosslinking targets Aldehydes (RCHO) and ketones (RCOR´) are reactive varieties of the more general functional group called carbonyls, which have a carbon–oxygen double bond (C=O). The polarity of this bond (especially in the context of aldehydes) makes the carbon atom electrophilic and reactive to nucleophiles such as primary amines. Although aldehydes do not naturally occur in proteins or other macromolecules of interest in typical biological samples, they can be created wherever oxidizable sugar groups (also called reducing sugars) exist. Such sugars are common monomer constituents of the polysaccharides or carbohydrates in posttranslational glycosylation of many proteins. In addition, the ribose of RNA is a reducing sugar. Periodic acid (HIO4 ) from dissolved sodium periodate (NaIO4 ) is a well-known mild agent for effectively oxidizing vicinal diols in carbohydrate sugars to yield reactive aldehyde groups. The carbon–carbon bond is cleaved between adjacent hydroxyl groups. By altering the amount of periodate used, aldehydes can be produced on a smaller or larger selection of sugar types. For example, treatment of glycoproteins with 1 mM periodate usually affects only sialic acid residues, which frequently occur at the ends of polysaccharide chains. At concentrations of 6–10 mM periodate, other sugar groups in proteins will be affected (Figure 10). Pyridyldithiol R S S N Pyridyldithiol reagent Cleavable conjugate (disulde bond) Sulfhydryl on protein R S S N SH + P pH 6.5–7.5 R S S P S N H + Pyridine-2-thione Figure 9. Pyridyldithiol reaction scheme for cleavable (reversible) chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the pyridyl disulfide reactive group. P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH). Internal mannose residue Terminal sialic acid residue Aldehyde-activated Aldehyde-activated O O O NH O OH O HO R O O O O O HO R´ R 10 mM 1 mM O O I O− O Na+ + Sodium meta-periodate 2 CH2 O O OH OH O O HO R´ R * O O OH OH OH NH O OH O HO R * * Figure 10. The reaction of sodium periodate with sugar residues yields aldehydes for conjugation reactions. R and R´ represent connecting sugar monomers of the polysaccharide. Red asterisks indicate sites of diol cleavage. Sialic acid is also called N-acetyl-Dneuraminic acid. 11 Hydrazides Carbonyls (aldehydes and ketones) can be produced in glycoproteins and other polysaccharide-containing molecules by mild oxidation of certain sugar glycols using sodium meta-periodate. Hydrazide-activated crosslinkers and labeling compounds will then conjugate with these carbonyls at pH 5–7, resulting in formation of hydrazone bonds. Hydrazide chemistry is useful for labeling, immobilizing, or conjugating glycoproteins through glycosylation sites, which are often (as with most polyclonal antibodies) located at domains away from the key binding sites whose function one wishes to preserve. Hydrazide reaction chemistry Aldehydes created by periodate oxidation of sugars in biological samples react with hydrazides at pH 5–7 to form hydrazone bonds (Figure 11). Although this bond to a hydrazide group is a type of Schiff base, it is considerably more stable than a Schiff base formed with a simple amine. The hydrazone bond is sufficiently stable for most protein-labeling applications. If desired, however, the double bond can be reduced to a more stable secondary amine bond using sodium cyanoborohydride (see section on reductive amination). Alkoxyamines Although not currently as popular or common as hydrazide reagents, alkoxyamine compounds conjugate to carbonyls (aldehydes and ketones) in much the same manner as hydrazides. Alkoxyamine reaction chemistry Alkoxyamine compounds conjugate to carbonyls to create an oxime linkage (Figure 12). The reaction is similar to that of hydrazides and can also use aniline as a catalyst. Reductive amination In reductive amination, the electrophilic carbon atom of an aldehyde attacks the nucleophilic nitrogen of a primary amine to yield a weak bond called a Schiff base bond. Unlike the bond formed with hydrazide or alkoxyamines, the Schiff base formed with ordinary amines rapidly hydrolyzes (reverses) in aqueous solution and must be reduced to an alkylamine (secondary amine) linkage to stabilize it. Sodium cyanoborohydride (NaCNBH3) is a mild reducing agent that performs this function effectively, without reducing other chemical groups in biological samples (Figure 13). Like carbodiimide crosslinking chemistry (carboxyl to amine), reductive amination (aldehyde to amine) is a zero-length crosslinking method. Hydrazide O NH2 N R H Hydrazide reagent Stable conjugate (hydrazone bond) Aldehyde (oxidized sugar) + O N N R H H P O NH2 N R H O H P pH 6.5–7.5 Figure 11. Hydrazide reaction scheme for chemical conjugation to an aldehyde. R represents a labeling reagent or one end of a crosslinker having the hydrazide reactive group. P represents a glycoprotein or other glycosylated molecule that contains the target functional group (i.e., an aldehyde formed by periodate oxidation of carbohydrate sugar groups, such as sialic acid). Alkoxyamine O NH2 R Alkoxyamine reagent Stable conjugate (oxime bond) Aldehyde (oxidized sugar) O NH2 R O N H R P O H + P pH 6.5–7.5 Figure 12. Alkoxyamine reaction scheme for chemical conjugation to an aldehyde. R represents a labeling reagent or one end of a crosslinker having the alkoxyamine reactive group. P represents a glycoprotein or other glycosylated molecule that contains the target functional group (i.e., an aldehyde formed by periodate oxidation of carbohydrate sugar groups, such as sialic acid). O O OH O O R O P O P O OH HO R O P N O O OH HO R O P NH P NH2 P Aldehyde groups (oxidized sugar groups) Conjugate (Schiff base bond) Stable conjugate (secondary amine bond) Primary amine NaCNBH3 Sodium cyanoborohydride Figure 13. Reductive amination, the conjugation of aldehydes and primary amines. The initial reaction results in a weak, reversible Schiff base linkage. Reduction with sodium cyanoborohydride creates a stable, irreversible secondary amine bond. 12 Photoreactive crosslinkers Photoreactive crosslinkers are widely used for nonspecific bioconjugation. While numerous options exist (Figure 14), the two most common photoreactive chemical groups are diazirines and aryl azides. Photoreactive groups are activated by ultraviolet (UV) light and can be used in vitro and in vivo. Aryl azides Photoreactive reagents are chemically inert compounds that become reactive when exposed to UV or visible light. Historically, aryl azides (also called phenyl azides) have been the most popular photoreactive chemical group used in crosslinking and labeling reagents. Photoreactive reagents are most often used as heterobifunctional crosslinkers to capture binding partner interactions. A purified bait protein is labeled with the crosslinker using the amine- or sulfhydryl-reactive end. Then this labeled protein is added to a lysate sample and allowed to bind its interactor. Finally, photoactivation with UV light initiates conjugation via the aryl azide group. Aryl azide reaction chemistry When an aryl azide is exposed to UV light (250–350 nm), it forms a nitrene group that can initiate addition reactions with double bonds, insertion into C–H and N–H sites, or subsequent ring expansion to react with a nucleophile (e.g., primary amines). The latter reaction path dominates when primary amines are present in the sample (Figure 15). Thiol-containing reducing agents (e.g., DTT or β-mercaptoethanol) must be avoided in the sample solution during all steps before and during photoactivation because they reduce the azide functional group to an amine, preventing photoactivation. Reactions can be performed in a variety of amine-free buffer conditions. If working with heterobifunctional photoreactive crosslinkers, buffers should be used that are compatible with both reactive chemistries involved. Experiments must be performed in subdued light and/or with reaction vessels covered in foil until photoreaction is intended. Typically, photoactivation is accomplished with a handheld UV lamp positioned close to the reaction solution and shining directly on it (i.e., not through glass or polypropylene) for several minutes. Three basic forms of aryl azides exist: simple phenyl azides, hydroxyphenyl azides, and nitrophenyl azides. Generally, short-wavelength UV light (e.g., 254 nm) is needed to efficiently activate simple phenyl azides, while long-wavelength UV light (e.g., 365 nm) is sufficient for nitrophenyl azides. Because short-wavelength UV light can be damaging to other molecules, nitrophenyl azides are usually preferable for crosslinking experiments. Diazirines Diazirines are a newer class of photoactivatable chemical groups that are being used in crosslinking and labeling reagents. The diazirine (azipentanoate) moiety has better photostability than aryl azide groups, and it is more easily and efficiently activated with long wavelength UV light (330–370 nm). Aryl azide N+ N N– R Aryl azide Tetrauorophenyl azide N+ N N− F F F F Diazirine N N N+ N N− Ortho-nitrophenyl azide Ortho-hydroxyphenyl azide OH N+ N N− O O N N+ N− Azido-methylcoumarin N+ N N− O N+ −O Meta-hydroxyphenyl azide Meta-nitrophenyl azide O O O Psoralen HO N+ N N− N+ N N− O N+ O− Figure 14. Common photoreactive chemical groups used for bioconjugation. N: H N R N H H N N R R R N N HN R N R Phenyl azide reagent Nitrene formed UV light Ring expansion Addition reactions Active hydrogen (N-H) insertion Active hydrogen (C-H) insertion Dihydroazepine intermediate N+ N N− Nucleophile R–NH2 R–H R–H Reactive hydrogen R–NH2 Figure 15. Aryl azide reaction scheme for light-activated photochemical conjugation. Squiggle bonds represent a labeling reagent or one end of a crosslinker having the phenyl azide reactive group. R represents a protein or other molecule that contains nucleophilic or active hydrogen groups. Bold arrows indicate the dominant pathway. Halogenated aryl azides react directly (without ring expansion) from the activated nitrene state. Diazirine R N N 13 Diazirine reaction chemistry Photoactivation of diazirine creates reactive carbene intermediates (Figure 16). Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent. Diazirine analogs of amino acids can be incorporated into protein structures by translation, enabling specific recombinant proteins to be activated as the crosslinker. Chemoselective ligation Chemoselective ligation refers to the use of mutually specific pairs of conjugation reagents. Unlike typical crosslinking methods used in biological research, this reaction chemistry depends upon a pair of unique reactive groups that are specific to one another and also foreign to biological systems. Because these reactions (azide–alkyne or azide–phosphine) do not occur in cells, these functional groups react only with each other in biological samples, thus resulting in minimal background and few artifacts, hence the term “chemoselective”. This specialized form of crosslinking can be applied for both in vivo metabolic labeling and bioconjugation using bioorthogonal coupling partners. Chemoselectivity of azide–alkyne reactions The reaction between an azide and an alkyne either using a copper catalyst or a copper-free strained alkyne results in the formation of a stable triazole linkage between the coupling partners. This reaction has received much attention because of the bioorthogonal nature of the two coupling partners. In the classic click reaction, an azide is coupled to an alkyne using Cu(I) to bring two coupling partners together and form a stable triazole linkage. One drawback of this approach is that copper ions—both Cu(II) and Cu(I), which are produced in the presence of ascorbate or TCEP—can harm cells, reduce the fluorescence of fluorophores, and impair protein function. To overcome this challenge, strained cyclic alkynes (DIBO/DBCO) have been developed to efficiently react with an azide to form the triazole linkage in the absence of copper under biological conditions. The strain in this eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts or extreme temperatures, enabling the study of the surface of live cells, and preventing copper-induced damage of fluorescent proteins such as GFP in fixed and permeabilized cells (Figure 17). Chemoselectivity of azide–phosphine reactions The Staudinger reaction occurs between a methyl ester phosphine and an azide (N3 – ) to produce an aza-ylide intermediate that is trapped to form a stable covalent bond (Figure 18). The Staudinger ligation involves the reaction of azido bioorthogonal probes with phosphine compounds. Similar to click chemistry, the ligation reaction is highly specific and can be performed in aqueous environments at physiological pH. Staudinger ligations do not require copper to be reactive, which increases their biocompatibility; however, these reactions also tend to be slower than click reactions because of the absence of a catalyst. Figure 17. Two-step reaction scheme for conjugating a protein R and azide containing coupling partner P with TFP-PEG(n)-DBCO. In this example, the TFP-PEG(n)-DBCO is first reacted with the protein to produce a DBCO-labeled protein. After excess nonreacted crosslinker and by-products are removed, the DBCO-labeled protein is reacted with the appropriate molar ratio of azide-coupling partner containing an azide group, forming a stable triazole linkage. Phosphine-activated compound OCH3 O O P Ph Ph H N R Azide-labeled target molecule N3 P N H O O P O Ph Ph H N R P H2O CH3OOCH3 O O P N Ph Ph H N P R Conjugate (amide bond) Aza-ylide intermediate Figure 18. Staudinger ligation reaction scheme (azide–phosphine conjugation). Phosphine-activated proteins or labeling reagents react with azide-labeled target molecules to form aza-ylide intermediates that quickly rearrange in aqueous conditions to form stable amide bonds between reactant molecules. Diazirine reagent Stable conjugate (insertion bond) R N N R P H R´ R´ P H N2 UV light 350 nm R : Carbene formed Figure 16. Diazirine reaction scheme for light-activated photochemical conjugation. R represents a labeling reagent or one end of a crosslinker having the diazirine reactive group. P represents a protein or other molecule that contains nucleophilic or active hydrogen groups (R´). Phosphine OCH3 O O P Ph Ph H N R Azide N3 P DBCO-labeled protein N3 Conjugated protein N O O H N O O O F F F F n n n P NH2 R H N N O O N H O N N N P R H N N O N H P O O O O 14 Molecular properties of crosslinking reagents Introduction 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 if 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 3). 15 Homobifunctional and heterobifunctional crosslinkers Crosslinkers can be classified as homobifunctional or heterobifunctional. Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm (Figure 19). Generally, they must be used in one-step reaction procedures to randomly “fix” or polymerize molecules containing like functional groups. For example, adding an amine-to-amine crosslinker to a cell lysate will result in random conjugation of protein subunits, interacting proteins, and any other polypeptides whose lysine side chains happen to be near each other in the solution. This is ideal for capturing a “snapshot” of all protein interactions but cannot provide the precision needed for other types of crosslinking applications. For example, when preparing an antibody–enzyme conjugate, the goal is to link one to several enzyme molecules to each molecule of antibody without causing any antibody-to-antibody linkages to form. This is not possible with homobifunctional crosslinkers. Heterobifunctional crosslinkers possess different reactive groups at either end (Figure 20). These reagents not only allow singlestep conjugation of molecules that have the respective target functional groups, but they also allow sequential (two-step) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heterobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess unreacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs. The most widely used heterobifunctional crosslinkers are those having an amine-reactive group (succinimidyl ester, NHS ester) at one end and a sulfhydryl-reactive group (e.g., maleimide) on the other end. Because the NHS ester group is less stable in aqueous solution, it is usually reacted to one protein first. If the second protein does not have available native sulfhydryl groups, they can be added in a separate prior step using sulfhydryl-addition reagents. Learn more at thermofisher.com/proteincrosslinking Table 3. Molecular properties of bioconjugation reagents. Property Description Chemical specificity The reactive target(s) of the crosslinker’s reactive ends. A general consideration is whether the reagent has the same or different reactive groups at either end (termed homobifunctional and heterobifunctional, respectively). General reaction conditions The buffer system required to perform bioconjugation. Variables include pH, buffer concentration, and protein concentration. Modifications These specialized reagents add molecular mass, create a new functional group that can be targeted in a subsequent reaction step, or increase the solubility of the molecule. Spacer arm length The molecular span of a crosslinker (i.e., the distance between conjugated molecules). A related consideration is whether the linkage is cleavable or reversible. Spacer arm composition The chemical groups found within the spacer arm. Spacer arm cleavability The availability of a cleavage site within the spacer arm between the chemical reactive groups. Chain structure and solubility The presence of a straight or branched chain that can effect whether a crosslinker or modifier can permeate into cells and/or crosslink hydrophobic proteins within membranes. These properties are determined by the composition of the spacer arm and/or reactive group. N O O O O O N O O O DSS Disuccinimidyl suberate MW 368.34 Spacer arm 11.4 Å Figure 19. Homobifunctional crosslinker example. DSS is a popular, simple crosslinker that has identical amine-reactive NHS ester groups at either end of a short spacer arm. The spacer arm length (11.4 Å) is the final maximum molecular distance between conjugated molecules (i.e., nitrogens of the target amines). Sulfo-SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate MW 436.37 Spacer arm 8.3 Å O O N O O N O O S Na+OO O Figure 20. Heterobifunctional crosslinker example. Sulfo-SMCC is a popular crosslinker that has an amine-reactive sulfo-NHS ester group (left) at one end and a sulfhydryl reactive maleimide group (right) at the opposite end of a cyclohexane spacer arm. This allows sequential, two-step conjugation procedures. 16 Certain reagents have three termini, and are referred to as trifunctional crosslinkers or label transfer reagents. These compounds typically possess two chemically reactive functional groups and one label such as a biotin group. A commonly used example is sulfo-SBED (Figure 21). General reaction conditions In many applications, it is necessary to maintain the native structure of the protein complex, so crosslinking is most often performed using near-physiological conditions. Reaction buffers for commonly used reactive groups are summarized in Table 4. Optimal crosslinker-to-protein molar ratios for reactions must be determined empirically, although product instructions for individual reagents generally contain guidelines and recommendations for common applications. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low to moderate degree of conjugation may be optimal so that biological activity of the protein is retained. The number of functional groups on the protein’s surface is also important to consider. If there are numerous target groups, a lower crosslinker-to-protein ratio can be used. For a limited number of potential targets, a higher crosslinker-to-protein ratio may be required. Furthermore, the number of components should be kept low or to a minimum because conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits. Modifications 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. Protein modification reagents are chemicals that block, add, change, or extend the molecular reach of functional groups. Protein sulfhydryls (side chain of cysteine) are important regulators of protein structure and function. Reducing agents are used to prevent intra- and intermolecular disulfide bonds Table 4. Summary of characteristics of reactive groups. Reactive group Target molecules Reaction buffer Optimal reaction pH range Amine Aldehydes (oxidized carbohydrates) Carboxylic acid (EDC-modified) PBS (non-amine) MES pH 7.2 pH 4.5–7.2 Aryl azide Unsubstituted aryl azides react primarily with amines PBS (non-amine) NA Carbodiimide Carboxylic acids, hydroxyls MES or PBS pH 4.5–7.2 Hydrazide Aldehydes, ketones Carboxylic acid (EDC-modified) 0.1 M Na-acetate/phosphate MES/non-amine buffer pH 5.5–7.5 pH 4.5–7.2 (up to pH 7.5) Imidoester Amines PBS, borate, carbonate/bicarbonate, HEPES pH 8–9 Iodoacetyl Sulfhydryls PBS, borate, carbonate/bicarbonate, HEPES pH 7.5–8.5 Isocyanate (PMPI) Hydroxyls Amines Nonaqueous NA Maleimide Sulfhydryls Thiol-free pH 7 optimal pH 6.5–7.5 NHS ester Amines PBS, borate, carbonate/bicarbonate, HEPES pH 7.5 optimal pH 7.2–8.5 Pyridyl disulfide Sulfhydryls Thiol-free pH 7–8 Vinyl sulfone (HBVS) Sulfhydryls Thiol-free pH 8 Sulfo-SBED MW 879.98 HN S S S O HN O H N O N O O O HN NH O N O N+ NS O O Na+O13.7 Å 9.1 Å 19.1 Å Figure 21. Trifunctional crosslinker example: sulfo-SBED. 17 from forming between cysteine residues of proteins in order to enable crosslinking or modification. This reduction is sometimes carried out under denaturing conditions to enhance reactivity of inaccessible disulfide bonds. Dithiothreitol (DTT), β-mercaptoethanol (BME), and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) are frequently used to reduce the disulfide bonds of proteins. TCEP-HCl is a potent, versatile, odorless, thiol-free reducing agent with broad application to protein and other research involving reduction of disulfide bonds (Figure 22). This unique compound is easily soluble and very stable in many aqueous solutions. TCEP reduces disulfide bonds as effectively as dithiothreitol (DTT), but unlike DTT and other thiol-containing reducing agents, TCEP does not have to be removed before certain sulfhydryl-reactive crosslinking reactions. Chaotropic and denaturing chemical agents, including urea and guanidine hydrochloride, disrupt water interactions and promote hydrophobic protein and peptide solubilization, elution, refolding, and structural analysis. Certain reagents are capable of reacting permanently or reversibly with sulfhydryl groups (e.g., NEM or MMTS, respectively). These reagents add a very small “cap” on the native sulfhydryl, enabling the activity of certain enzymes to be controlled for specific assay purposes (Figure 23). Sulfo-NHS acetate is a protein modification reagent that reacts with primary amines at pH 7.0–9.0, allowing modifications to occur in many standard buffers without amines (Figure 24). Once reacted, the amine is irreversibly capped with an acyl group. For reversible amine blocking, Thermo Scientific™ Pierce™ Citraconic Anhydride (Cat. No. 15479100) is used. SulfoNHS acetate is typically used to prevent polymerization when performing protein crosslinking reactions and when conjugating peptides to carrier proteins for immunogen production. Blocking amines on the peptide allows directed conjugation of carboxylic acids on the peptide to primary amines on the protein using Thermo Scientific™ EDC (Cat. No. 22980, 22981). SATA and related reagents contain an amine-reactive group and a protected sulfhydryl group. By reacting the compound to a purified protein, the side chain of lysine residues can be modified to contain a sulfhydryl group for targeting with sulfhydryl-specific crosslinkers or immobilization chemistries. The method does not actually convert the amine into a sulfhydryl; rather, it attaches a sulfhydryl-containing group to the primary amine. The effect is also to extend the length of the side chain by several angstroms (Figure 25). Chemically attaching single- or branched-chain polyethylene glycol (PEG) groups to proteins (often referred to as PEGylation) is a form of labeling or modification that is primarily used to confer water solubility and inert molecular mass to proteins. Forms of PEG that have been synthesized to contain reactive chemical groups comprise ready-to-use, activated reagents for PEGylation (Figure 26). Learn more at thermofisher.com/proteinmodification H3 C S O S CH3 O MMTS Methyl methanethiosulfonate MW 126.20 O O CH3 N NEM N-Ethylmaleimide MW 125.13 Figure 23. Sulfhydryls can be blocked using NEM and MMTS. O O O P OH HO OH O O O O HO P OH R OH S R S R R HS SH O + + H H + TCEP Figure 22. TCEP reduces disulfide bonds within bioconjugation reagents and proteins. O N O O O S Na +O– O O Sulfo-NHS acetate MW 259.17 Figure 24. Sulfo-NHS acetate is a protein modification reagent for blocking primary amines. N O O O O S O SATA MW 231.23 Spacer arm 2.8 Å Traut’s reagent MW 137.63 Spacer arm 8.1 Å S NH2 +Cl – Figure 25. Sulfhydryls can be converted to amines using SATA or Traut’s reagent. 18 Spacer arm length The spacer arm is the chemical chain between two reactive groups or between a reactive group and a label. The length of a spacer arm (measured in Å) determines how flexible a conjugate will be. Longer spacer arms have greater flexibility and reduced steric hindrance. Longer spacer arms have the caveat of possessing more sites for potential nonspecific binding. Spacer arms can range from zero length to >100 Å (Figure 27). Spacer arm composition The molecular composition of a bioconjugation spacer arm can affect solubility and nonspecific binding. Traditional crosslinkers and labeling reagents have spacer arms that contain hydrocarbon chains or polyethylene glycol (PEG) chains. Hydrocarbon chains are not water-soluble and typically require an organic solvent such as DMSO or DMF for suspension. These reagents are better suited for penetrating the cell membrane and performing intercellular crosslinking because they are hydrophobic and uncharged. For example, if a charged sulfonate group is added to the termini of a hydrophobic crosslinker, a water-soluble analog is formed. A good example of this is the comparison of DSS with BS3 . DSS is soluble in organic solvents whereas BS3 is soluble is aqueous buffers. BS(PEG)5 is also water-soluble because of its PEG spacer (Figure 28). Spacer arm cleavability Crosslinkers and protein modification reagents form stable, covalent bonds with the proteins they react with. In certain applications, it is desirable to have the ability to break that bond and recover the individual components. Bioconjugation reagents are available with a cleavage site built into the spacer arm. The most commonly used cleavage site is a disulfide bridge, which can be readily reduced with the introduction of a common reducing agent such as β-mercaptoethanol, dithiothreitol, or TCEP. An example of this is the crosslinker DTBP (Figure 29). DTBP Dimethyl 3,3' dithiobispropionimidate·2HCl MW 309.28 Spacer arm 11.9 Å S S O O NH2 Cl– NH2 Cl– + + Figure 29. The disulfide bridge built into the spacer arm of DTBP allows easy cleavage of a protein conjugate using standard reducing agents. MS(PEG)4 MW 333.33 Spacer arm 16.4 Å n = 8 MS(PEG)8 MW 509.4 Spacer arm 30.8 Å n = 12 MS(PEG)12 MW 685.71 Spacer arm 44.9 Å n = 24 MS(PEG)24 MW 1,214.39 Spacer arm 88.2 Å MS(PEG)n Methyl-PEGn -NHS ester Succinimidyl-((N-methyl)-n(ethylene glycol)) ester O O O N O O CH3 n O O O O O N O O O CH3 16.4 Å Figure 26. Examples of single-chain, amine-reactive PEGylation reagents. DFDNB 1,5-diuoro-2,4-dinitrobenzene MW 204.09 Spacer arm 3.0 Å N + O– O N – O + O F F N O O O O O N O O O DSS Disuccinimidyl suberate MW 368.34 Spacer arm 11.4 Å BS(PEG)9 Bis(succinimidyl) nona(ethylene glycol) MW 708.71 Spacer arm 35.8 Å N O O O O O O O O O N O O O O O O O O Figure 27. Different lengths of spacer arms. N O O O O O N O O O DSS Disuccinimidyl suberate MW 368.34 Spacer arm 11.4 Å BS3 Bis(sulfosuccinimidyl) suberate MW 572.43 Spacer arm 11.4 Å N O O O O O N O O O S O O S O O Na+O– O–Na+ N O O O O O O O O O N O O O O BS(PEG)5 Bis(succinimidyl) penta(ethylene glycol) MW 532.50 Spacer arm 21.7 Å Figure 28. Crosslinkers with various spacer arm compositions. 19 Spacer arm structure and solubility Crosslinkers typically possess a straight chain spacer arm, but protein modification reagents allow more options. A good example is Thermo Scientific™ PEGylation reagents that can be either straight or branched. For example, CA(PEG)n is a straight-chain PEGylation reagent and TMM(PEG)12 is a branched reagent (Figure 30). Many crosslinkers, by virtue of their hydrophobic spacer arms, have limited solubility in aqueous solutions. These crosslinkers are generally dissolved in DMF or DMSO, then added to the biological system or solution of biomolecules to be crosslinked. Hydrophobic crosslinkers are able to cross cellular and organellar membranes and affect crosslinking both at the outer surface of a membrane and within the membrane-bound space. It is often inconvenient or undesirable to introduce organic solvents into a crosslinking procedure for a biological system. It is also desirable in many instances to affect crosslinking only on the outer surface of a cellular or organellar membrane without altering the interior of the cell or organelles. For such cases, several water-soluble, membrane-impermeant crosslinkers are available. Some crosslinkers contain a spacer arm formed from PEG subunits, resulting in a polyethylene oxide (PEO) chain with abundant oxygen atoms to provide water solubility. These crosslinkers are designated by a (PEG)n in their name and are both water-soluble and unable to penetrate biological membranes. They provide the added benefit of transferring their hydrophilic spacer to the crosslinked complex, decreasing the potential for aggregation and precipitation of the complex. Other crosslinkers obtain their water solubility and their ability to permeate the membrane by virtue of a charged reactive group at either end of the spacer. These charged reactive groups, such as sulfo-NHS esters or imidoesters, impart water solubility to the crosslinking reagent, but not to the crosslinked complex because the reactive group is not a part of the final complex. Figure 30. Straight and branched protein modification reagents. HN O O O O H N N H O O O N H O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O CH3 CH3 CH3 HN O O O O N O O O TMM(PEG)12 (Methyl-PEG12) 3 -PEG4 -maleimide MW 2,360.75 46.2 Å 52.0 Å 27.6 Å CA(PEG)4 MW 265.30 Spacer arm 18.1Å O O O O O HO NH2 CA(PEG)n Carboxy-PEGn -amine Carboxyl-(ethyleneglycol)n ethylamine 18.1Å 20 Applications using bioconjugation and crosslinking reagents Introduction Bioconjugation and crosslinking reagents have a variety of applications in life science research and assay development. These include protein and peptide biotinylation, antibody labeling with fluorophores or drugs, protein immobilization onto solid supports, protein–protein conjugation, label transfer, protein interaction and crosslinking using mass spectrometry, in vivo crosslinking, metabolic labeling, and cell membrane structural studies. Commonly, antibodies are the target of bioconjugation with applications in purification and detection in a complex biological sample. In this process, NHS ester chemistry is the most widely used method for labeling available lysine residues. 21 Protein and peptide biotinylation The highly specific interaction of avidin with biotin (vitamin H) can be a useful tool in designing nonradioactive purification and detection systems. The extraordinary affinity of avidin for biotin (Ka = 1015 M–1) is the strongest known noncovalent interaction of a protein and ligand, and allows biotin-containing molecules in a complex mixture to be discretely bound with avidin conjugates. Desthiobiotin is a modified form of biotin that binds less tightly to avidin and streptavidin than biotin while still providing excellent specificity in affinity purification methods. Unlike biomolecules that are labeled with biotin, proteins and other targets that are labeled with desthiobiotin can be eluted without harsh denaturing conditions (Figure 31). The extensive line of Thermo Scientific™ biotinylation labeling reagents exploits this unique interaction. Biotin, a 244 Da vitamin found in tiny amounts in all living cells, binds with high affinity to avidin-based molecules. Since biotin is a relatively small molecule, it can be conjugated to many proteins without significantly altering their biological activity. The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules. Using these reactive groups, biotin can be easily attached to most proteins and peptides. Biotinylation reagents are available for targeting a variety of functional groups, including primary amines, sulfhydryls, carbohydrates, and carboxyls. Photoreactive biotin compounds that react nonspecifically upon photoactivation are also available. This variety of functional group specificities is extremely useful, allowing the choice of a biotinylation reagent that does not inactivate the target macromolecule. Several cleavable or reversible biotinylation reagents are also available and allow specific elution of the biotinylated molecule from biotin-binding proteins and peptides. The most frequently used biotinylation reagents, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfo-NHS) esters, react with primary amines (Figure 32). While NHS esters of biotin are the most frequently used biotinylation reagents, they are not necessarily the best for a particular application. If only a portion of the primary amines on a protein are reacted, reaction with NHS esters of biotin will result in a random distribution of biotin on the surface of the protein. If a particular primary amine is critical to the biological activity of the protein, modification of this critical amine may result in the loss of its biological activity. Depending on the extent of biotinylation, complete loss of activity may occur. Antibodies are biotinylated more often than any other class of proteins, and it is advantageous to biotinylate in a manner that will maintain immunological reactivity. Thermo Scientific™ Sulfo-NHS-LC-Biotin is an excellent choice for labeling both monoclonal and polyclonal antibodies because it is the simplest and often the most effective method. Remove sulfur Biotin MW 244.31 Desthiobiotin MW 214.26 O O S HN NH HO O O HN NH HO Figure 31. Comparison of the chemical structures of biotin and desthiobiotin. Sulfo-NHS-LC-biotin O O H N O NH HN S O N O O S O– O O Sulfo-NHS leaving group (remove by desalting) Biotinylated molecule Protein with primary amines NH2 P OH N O O S O– O O N H O H N O NH HN S O P Figure 32. Biotinylation using NHS ester chemistry. 22 Antibody labeling and crosslinking Antibodies, like other proteins, can be covalently modified in many ways to suit the purpose of an assay. Many immunological methods involve the use of labeled antibodies and a variety of reagents have been created to allow labeling of antibodies. Enzymes, biotin, fluorophores, and various small molecules are all commonly used to provide a detection signal in biological assays. The most common target for antibody labeling or conjugation is primary amines, which are found primarily on lysine residues. They are abundant, widely distributed, and easily modified because of their reactivity and their location on the surface of the antibody. Primary amines can be targeted using several kinds of conjugation chemistries. The most commonly used reagent for primary amine labeling is the N-hydroxysuccinimidyl ester (NHS ester) reactive group. Many reactive crosslinkers, fluorescent labeling products, or biotinylation reagents are commercially available pre-activated with an NHS-ester group. Understanding the functional groups available on an antibody is the key to determining a strategy for modification. • Primary amine groups (–NH2 ) are found on lysine side chains and at the amino terminus of each polypeptide chain. • Sulfhydryl groups (–SH) can be generated by reducing disulfide bonds in the hinge region. • Carbohydrate residues containing cis-diols can be oxidized to create active aldehydes (–CHO). In any particular antibody clone, lysines (primary amines) might occur prominently within the antigen binding site. Thus, the lone drawback to this labeling strategy is that it occasionally causes a significant decrease in the antigen-binding activity of the antibody. The decrease may be particularly pronounced when working with monoclonal antibodies or when attempting to add a high density of labels per antibody molecule. A simple alternative to these traditional methods is the Invitrogen™ SiteClick™ antibody labeling system (Figure 33), which allows simple and gentle site-selective attachment of compounds to the carbohydrate domains present only on the heavy chains of essentially all IgG antibodies regardless of isotype and host species. This method also provides excellent reproducibility from labeling to labeling and from antibody to antibody because the N-linked glycans are highly conserved. The Invitrogen™ SiteClick™ Antibody Azido Modification Kit uses enzymes to specifically attach azido moieties to the antibody carbohydrate domains. Once the azide is attached, a variety of sDIBO alkyne labels are available to conjugate with a simple incubation step. A number of different conjugates can be site-selectively attached to the heavy chain glycans—including Invitrogen™ Alexa Fluor™ dyes, pHrodo™ dyes, R-PE, biotin, Invitrogen Qdot™ probes, or independently supplied DIBO/DBCO conjugates. Figure 33. The SiteClick antibody labeling system. The first step in the SiteClick antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor™ 488 DIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody. Antibody • Polyclonal • Monoclonal • Recombinant Azide-activated antibody N2 N2 N3 N2 Site-selectively labeled antibody DIBO alkyne label β-Galactosidase GalT(Y269L) UDP-GalNAz Learn more at thermofisher.com/siteclick 23 Synthesis of antibody–drug conjugates Over the past decade, biologics have been increasingly pursued as therapeutic agents. In the case of antibody–drug conjugates (ADCs), internalization of the antibody is a powerful mechanism of action. Internalization moves the ADC from its binding site at the plasma membrane of the target cell to the lytic environment of a lysosome, resulting in activation of the attached toxin (Figure 34). The efficiency of this internalization process is directly linked to the therapeutic index of the ADC. We have combined powerful pHrodo dyes and SiteClick antibody labeling technologies to provide easy-to-use antibody labeling tools for creating antibody conjugates and studying internalization. Biomolecules labeled with pHrodo dyes are essentially nonfluorescent outside of cells at neutral pH and become brightly fluorescent in the acidic environment of lysosomes after they are internalized. This feature enables a no-wash, no-quench assay for detection of endocytosis, trafficking of ADCs in live cells, and cell killing by the antibody–drug conjugate (Figure 35). Fluorescent signals are only detectable for antibodies that have been specifically internalized. In addition, the location of the internalized antibody in the endocytic pathway can be studied. Figure 35. Cell killing with an antibody–drug conjugate. (A) Trastuzumab, a therapeutic antibody targeting HER2, was labeled with Invitrogen™ pHrodo™ iFL Red dye using SiteClick conjugation. A spheroid of SKBR3 (HER2+ ) breast cancer cells was treated for 48 hours with 30 nM of the labeled trastuzumab and Invitrogen™ CellEvent™ Caspase-3/7 Green sensor. Live-cell imaging was performed on the Thermo Scientific™ CellInsight™ CX7 HCA Platform. Red fluorescence indicates antibody delivery specifically into HER2+ cells. (B) Trastuzumab was labeled with both pHrodo iFL Red dye and the tubulin-destabilizing drug MMAE, using SiteClick conjugation. An SKBR3 (HER2+ ) spheroid was treated for 48 hours with 30 nM of the labeled antibody–drug conjugate and CellEvent Caspase-3/7 Green sensor. Live-cell imaging was performed on the CellInsight CX7 platform. Red fluorescence indicates antibody delivery specifically into HER2+ cells. Green indicates cell killing by the antibody–drug conjugate. Figure 34. Internalization of an antibody–drug conjugate. An antibody–drug conjugate (ADC) comprises a monoclonal antibody directed, for example, against a tumor cell antigen, coupled to a small cytotoxic molecule. An ADC is designed to specifically bind to target cells, where it is rapidly internalized. Typically the drug is liberated following trafficking to the lysosome, resulting in a highly targeted chemotherapeutic agent. Trastuzumab SiteClick pHrodo iFL Red/MMAE conjugate CellEvent Caspase-3/7 Green Detection Reagent Trastuzumab SiteClick pHrodo iFL Red conjugate CellEvent Caspase-3/7 Green Detection Reagent pHrodo iFL Red sDIBO pHrodo iFL Red sDIBO MMAE sDIBO pHrodo iFL Red sDIBO Binding Internalization Toxin release 24 Protein immobilization onto solid supports Proteins, peptides, and other molecules can be immobilized onto solid supports for affinity purification of proteins or for sample analysis. The supports may be nitrocellulose or other membrane materials, polystyrene plates or beads, agarose, beaded polymers, or glass slides. Some supports can be activated for direct coupling to a ligand. Other supports are made with nucleophiles or other functional groups that can be linked to proteins using crosslinkers. Carbodiimides such as Thermo Scientific™ EDC (Cat. No. 22980, 22981) are very useful for coupling proteins to carboxy- and amine-activated glass, plastic, and agarose supports. Carbodiimide procedures are usually one-step methods; however, two-step methods are possible if reactions are performed in organic solvents, or if Thermo Scientific™ NHS (Cat. No. 24500) or Sulfo-NHS (Cat. No. 24510) is used to enhance the reaction. EDC is useful for coupling ligands to solid supports and to attach leashes onto affinity supports for subsequent coupling of ligands. Useful spacers are diaminodipropylamine (DADPA), ethylenediamine, hexanediamine, 6-aminocaproic acid, and any of several amino acids or peptides. Spacer arms help to overcome steric effects when the ligand is immobilized too close to the matrix to allow access by the receptor. Steric effects are usually most pronounced when the ligand is a small molecule. The aldehydeactivated Thermo Scientific™ AminoLink™ Plus agarose resin (Cat. No. 20501) uses reductive amination, Thermo Scientific™ Pierce™ NHS-activated agarose (Cat. No. 26200) uses NHS ester chemistry, and Thermo Scientific™ SulfoLink™ resin (Cat. No. 20401) uses haloacetyl chemistry to immobilize molecules, whereas Thermo Scientific™ CarboxyLink™ resin (Cat. No. 20266) uses carbodiimide chemistry (Figure 36). Thermo Scientific™ crosslinkers DMP (Cat. No. 21666) and DSS (Cat. No. 21555) are used to immobilize antibodies on protein A or protein G supports for antigen purification. After the antibody binds to the Fc-binding proteins, the antibody is oriented so that the Fab region is available for antigen binding. DSS or DMP is applied to the bound antibody column to link the two proteins through primary amines. Thermo Scientific™ Pierce™ Crosslink IP Kit (Cat. No. 26147) is based on this chemistry and utilizes DSS to covalently immobilize the captured antibody to protein A/G agarose resin (Figure 37). The antibody resin is then incubated with the sample that contains the protein antigen of interest, allowing the antibody–antigen complex to form. After washing, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit. The entire procedure is performed in a microcentrifuge spin cup, allowing solutions to be fully separated from the agarose resin upon brief centrifugation. Only the antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments. Furthermore, the antibody resin often can be reused for additional rounds of immunoprecipitation. Figure 36. Immobilization of biomolecules onto solid supports using different bioconjugation chemistries. DSS crosslinker Agarose Bead Agarose Bead Protein A/G Protein A/G Agarose bead Agarose bead Figure 37. Covalent attachment of captured antibody to protein A/G agarose resin using the crosslinker DSS. EDC crosslinker Covalently immobilized ligand (attached by amide bond and long spacer arm) Immobilized DADPA (CarboxyLink Resin) + Carboxyl ligand (e.g., peptide C-terminus) O HO Peptide H N H N NH2 Agarose bead Peptide Agarose bead H N H N H N O + HS Peptide SulfoLink coupling resin Agarose bead H N I O 12-atom spacer Iodoacetyl group Peptide Agarose bead H N S O Thioether bond + HI Reduced sulfhydryl molecule Immobilized peptide + Amine ligand (antibody) Y Y Bead Y Y Y Y Y Covalently immobilized antibody NHS-activated agarose resin Agarose bead O O N O O NH2 NH2 H2 N H2 N + NH2 NH2 H2 N H2 N Amine ligand (antibody) Y Y Bead Y Y Y Y Y Covalently immobilized antibody Aldehyde-activated agarose resin Agarose bead C O H 25 Surface modification using PEG-based reagents Polyethylene glycol (PEG) compounds of discrete chain length can provide linkers of known molecular dimension for creating biocompatible planar surfaces or particles. In particular, PEG reagents containing a carboxylate group on one end and a thiol or lipoamide group on the other are effective as hydrophilic bridges between an adsorptive surface and an affinity ligand. Combining CA(PEG)12 molecules with MA(PEG)8 derivatives (or CT(PEG)12 with MT(PEG)8 as thiol reagents) in surface modification can form a hydrophilic lawn of methyl ether–terminated PEGs with periodic exposed carboxylic acid–containing PEGs (Figure 38). The exposed carboxylic acid groups can be coupled to affinity ligands using the carbodiimide coupling reaction with EDC and sulfo-NHS. In addition, functionalization of solid surfaces with polyethylene glycol spacers significantly reduces nonspecific protein binding. Thiols readily bind to gold surfaces, forming dative bonds, and have been used extensively for the modification of various surfaces such as quantum dots, self-assembled monolayers, and magnetic particles. Monodentate thiols, however, can be easily removed by compounds such as DTT. Bidentate thiols, such as lipoamides, provide an added level of stability to the nanostructure and are much more resistant to removal from the metal surface by DTT or similar reagents. In addition to their increased stability, bidentate thiols provide added flexibility owing to the fact that they can be used with a variety of metal surfaces (e.g., gold or silver nanoparticles). Learn more at thermofisher.com/pegylation O OH Surface or particle containing carboxylates Amine-reactive sulfo-NHS esters PEGylated surface containing a “lawn” of mPEG molecules with longer carboxy-PEG chains sticking o for subsequent conjugation with amine-containing ligands EDC/sulfo-NHS Methyl-PEG8 -amine Amino-PEG12-carboxylate O O S O OO O O N O HO O O O O O O O O O O HN O O H3 C O O O O O O O NH O O H3 C O O O O O O O NH O O H3 C O O O O O O O NH O O H3 C O O O O O O O NH O O O O HO O O O O O O O O O O HN O O O Figure 38. Shorter methyl-PEGn-amine reagents can be combined with longer PEG compounds containing terminal carboxylate groups to create the classic “flowers in the grass” surface modification. The carboxylate groups then can be used to immobilize affinity groups using a carbodiimide coupling procedure with EDC and sulfo-NHS. 26 Hapten–carrier conjugation for antibody production Several approaches are available for conjugating haptens to carrier proteins. The choice of which conjugation chemistry to use depends on the functional groups available on the hapten, the required hapten orientation and distance from the carrier, and the possible effect of conjugation on biological and antigenic properties. For example, proteins and peptides have primary amines (the N terminus and the side chain of lysine residues), carboxylic groups (the C terminus and the side chain of aspartic acid and glutamic acid), and sulfhydryls (the side chain of cysteine residues) that can be targeted for conjugation. Generally, it is the many primary amines in a carrier protein that are used to couple haptens via a crosslinking reagent. Many crosslinkers are used for making conjugates for use as immunogens. The best crosslinker to use depends on the functional groups present on the hapten and the ability of the hapten–carrier conjugate to function successfully as an immunogen after its injection. Carbodiimides are good choices for producing peptide–carrier protein conjugates because both proteins and peptides usually contain several carboxyls and primary amines. Carbodiimides such as EDC react with carboxyls first to yield highly reactive unstable intermediates that can then couple to primary amines (Figure 39). This efficient reaction produces a conjugated immunogen in less than 2 hours. Often peptides are synthesized with terminal cysteines to enable attachment to supports or to carrier proteins using sulfhydryl- and amine-reactive, heterobifunctional crosslinkers. By attaching the crosslinker first to the carrier protein (with its numerous amines) and then to a peptide containing a reduced terminal cysteine, all peptide molecules can be conjugated with the same predictable orientation (Figure 40). This method can be very efficient and yield an immunogen that is capable of eliciting a good response upon injection. Learn more at thermofisher.com/carrierproteins Maleimide-activated carrier protein Carrier-peptide conjugate Sulfo-SMCC crosslinker NH2 Carrier protein NH O N O O Carrier protein O O N O O N O O S O– O O Na+ NH O N O O S Carrier protein Peptide HS Peptide Carboxylic acid EDC O-acylisourea active ester Peptide-carrier conjugate + N N C H N+ Cl– + Primary amine Isourea by-product O NH NH H N+ OH O C H2 N P N H O P C O O NH N H N+ C Figure 39. EDC-mediated conjugation of peptides and carrier proteins. Carrier proteins (C) and peptides (P) have both carboxyls and amines, so conjugation occurs in both orientations. Carrier proteins are very large in comparison to typical peptide haptens; therefore, numerous conjugation sites exist on each carrier protein molecule. Figure 40. Peptide conjugation to carrier proteins for antibody production. 27 Protein–protein conjugation One of the most common applications for crosslinkers is the production of protein–protein conjugates. Conjugates are often prepared by attachment of an enzyme, fluorophore, or other molecule to a protein that has affinity for one of the components in the biological system being studied. Antibody–enzyme conjugates (primary or secondary antibodies) are among the most common protein–protein conjugates used. Although secondary antibody conjugates are available and relatively inexpensive, enzyme-labeled primary antibodies are usually expensive and can be difficult to obtain. Many reagents are used for the production of antibody–enzyme conjugates. Glutaraldehyde conjugates are easy to make, but they often yield conjugates that produce high background in immunoassays. Carbohydrate moieties can be oxidized and then coupled to primary amines on enzymes in a procedure called reductive alkylation or amination. These conjugates often result in less background in enzyme immunoassays and are relatively easy to prepare; however, some self-conjugation of the antibody may occur (Figure 41). Homobifunctional NHS ester or imidoester crosslinkers may be used in a one-step protocol, but polymerization and self-conjugation are also likely. Homobifunctional sulfhydryl-reactive crosslinkers such as Thermo Scientific™ Pierce™ BMH (Cat. No. 22330) may be useful if both proteins to be conjugated contain sulfhydryls. Heterobifunctional crosslinkers are perhaps the best choices for antibody–enzyme or other protein–protein crosslinking. Unwanted selfconjugation inherent when using homobifunctional NHS ester reagents or glutaraldehyde can be avoided by using a Thermo Scientific™ Pierce™ reagent such as SMCC (Cat. No. 22360) or Sulfo-SMCC (Cat. No. 22322). Sulfo-SMCC is first conjugated to one protein, and the second is thiolated with SATA (Cat. No. 26102) or Traut’s reagent (Cat. No. 26101), followed by conjugation (Figure 42). Alternatively, disulfides in the protein may be reduced, and the two activated proteins are incubated together to form conjugates free of dimers of either protein. Any of the other NHS ester, maleimide, or pyridyl disulfide crosslinkers can be substituted for sulfo-SMCC in this reaction scheme. Heterobifunctional photoactivatable phenyl azide crosslinkers are seldom used for making protein–protein conjugates because of low conjugation efficiencies. Figure 42. Reaction scheme for labeling reduced antibody fragments with maleimide-activated enzymes. Figure 41. Reaction scheme for labeling antibodies with enzymes such as HRP using reductive amination. NaIO4 NaCNBH3 O O OH HO HO R O HRP O O OH O O R O HRP O O OH HO R O Ab N HRP O O OH HO R O Ab NH HRP NH2 Ab Glycoprotein sugars Aldehyde groups Conjugate (Schiff base bond) Stable conjugate (amine bond) Sodium meta-periodate oxidizes glycoprotein sugars to reactive aldehyde groups Protein primary amines react with aldehydes to form Schiff bases Sodium cyanoborohydride reduces Schiff bases to yield stable amine bonds Maleimide-activated enzyme Sulfo-SMCC crosslinker Enzyme–antibody conjugate NH O N O O Enz O O N O O N O O S O– O O Na+ NH O N O O S Enz Ab NH HS Ab 2 Enz Enzyme Sulfhydryl-activated antibody 28 Synthesis of immunotoxins Specific antibodies can be covalently linked to toxic molecules and then used to target antigens on cells. Often these antibodies are specific for tumor-associated antigens. Immunotoxins are brought into the cell by surface antigens and, once internalized, they proceed to kill the cell by ribosome inactivation or other means. The type of crosslinker used to make an immunotoxin can affect its ability to locate and kill the appropriate cells. For immunotoxins to be effective, the conjugate must be stable in vivo. In addition, once the immunotoxin reaches its target, the antibody must be separable from the toxin to allow the toxin to kill the cell. Thiol-cleavable, disulfide-containing conjugates have been shown to be more cytotoxic to tumor cells than noncleavable conjugates of ricin A immunotoxins. Cells are able to break the disulfide bond in the crosslinker, releasing the toxin within the targeted cell. Thermo Scientific™ Pierce™ SPDP (Cat. No. 21857) is a reversible NHS ester, pyridyl disulfide crosslinker used to conjugate aminecontaining molecules to sulfhydryls. For several years, this has been the “workhorse” crosslinker for production of immunotoxins. The amine-reactive NHS ester is usually reacted with the antibody first. In general, toxins do not contain surface sulfhydryls; therefore, sulfhydryls must be introduced into them by reduction of disulfides, which is common for procedures involving ricin A chain and abrin A chain, or through chemical modification reagents. A second SPDP molecule can be used for this purpose and is reacted with amines on the immunotoxin, then reduced to yield sulfhydryls. Another chemical modification reagent that is commonly used for production of immunotoxins is Thermo Scientific™ Pierce™ 2-Iminothiolane, also known as Traut’s reagent (Cat. No. 26101). Traut’s reagent reacts with amines and yields a sulfhydryl when its ring structure opens during the reaction. Label transfer Label transfer involves crosslinking interacting molecules (i.e., bait and prey proteins) with a labeled crosslinking agent and then cleaving the linkage between bait and prey such that the label remains attached to the prey. This method allows a label to be transferred from a known protein to an unknown interacting protein. The label can then be used to purify and/or detect the interacting protein. Label transfer is particularly valuable because of its ability to identify proteins that interact weakly or transiently with the protein of interest. New non-isotopic reagents and methods continue to make this technique more accessible and simple to perform by any researcher. Label transfer reagents can also have biotin built into their structure. This type of design allows the transfer of a biotin tag to an interacting protein after cleavage of a cross-bridge. Thermo Scientific™ Pierce™ Sulfo-SBED reagent (Cat. No. 33033) is an example of such a trifunctional reagent (see Figure 21, page 16). It contains an amine-reactive sulfo-NHS ester on one arm (built off the α-carboxylate of the lysine core), a photoreactive phenyl azide group on the other side (synthesized from the α-amine), and a biotin handle (connected to the ε-amino group of lysine). The arm containing the sulfo-NHS ester has a cleavable disulfide bond, which permits transfer of the biotin component to any captured proteins. In use, a bait protein first is derivatized with sulfo-SBED through its amine groups, and the modified protein is allowed to interact with a sample. Exposure to UV light (300–366 nm) couples the photoreactive end to the nearest available C–H or N–H bond in the bait–prey complex, resulting in covalent crosslinks between bait and prey. Upon reduction and cleavage of the disulfide spacer arm, the biotin handle remains attached to the protein(s) that interacted with the bait protein, facilitating isolation or identification of the unknown species using streptavidin, Thermo Scientific™ NeutrAvidin™ protein, or monomeric avidin. The architecture of this trifunctional label transfer reagent differs substantially from the bifunctional counterparts discussed above. The advantages become almost immediately apparent just by examining the structure. The reactive moieties are well-segregated within sulfo-SBED. Most importantly, with a biotin label designed into sulfo-SBED, radiolabeling with 125I is no longer necessary. The biotin label can be used to significant advantage in a label transfer application. For example, biotin can operate as a handle for purification of the prey protein or prey protein fragments or as a detection target using streptavidin-HRP and colorimetric or chemiluminescent substrates. Subunit crosslinking and protein structural studies Crosslinkers can be used to study the structure and composition of proteins in samples. Some proteins are difficult to study because they exist in different conformations with varying pH or salt conditions. One way to avoid conformational changes is to crosslink subunits. Amine-, carboxyl-, or sulfhydryl-reactive reagents are used for identification of particular amino acids or for determination of the number, location, and size of subunits. Short- to medium-length spacer arm crosslinkers are selected when intramolecular crosslinking is desired. If 29 the spacer arm is too long, intermolecular crosslinking can occur. Carbodiimides that result in no spacer arm, along with short-length conjugating reagents, such as amine-reactive Thermo Scientific™ Pierce™ DFDNB (Cat. No. 21525), can crosslink subunits without crosslinking to extraneous molecules if used in optimal concentrations and conditions (Figure 43). Slightly longer crosslinkers, such as Thermo Scientific™ DMP (Cat. No. 21666, 21667), can also crosslink subunits, but they may result in intermolecular coupling. Adjusting the reagent amount and protein concentration can control intermolecular crosslinking. Dilute protein solutions and high concentrations of crosslinker favor intramolecular crosslinking when homobifunctional crosslinkers are used. For determination or confirmation of the three-dimensional structure, cleavable crosslinkers with increasing spacer arm lengths may be used to determine the distance between subunits. Experiments using crosslinkers with different reactive groups may indicate the locations of specific amino acids. Once conjugated, the proteins are subjected to two-dimensional electrophoresis. In the first dimension, the proteins are separated using nonreducing conditions and the molecular weights are recorded. Some subunits may not be crosslinked and will separate according to their individual molecular weights, while conjugated subunits will separate according to the combined size. The second dimension of the gel is then performed using conditions to cleave the crosslinked subunits. The individual molecular weights of the crosslinked subunits can be determined. Crosslinked subunits that were not reduced will produce a diagonal pattern, but the cleaved subunits will be off the diagonal. The molecular weights of the individual subunits should be compared with predetermined molecular weights of the protein subunits using reducing SDS-polyacrylamide gel electrophoresis. Figure 43. DFDNB and DMP are used for crosslinking between protein subunits. DMP Dimethyl pimelimidate·2HCl MW 259.17 Spacer arm 9.2 Å O O NH2 +Cl– NH2 +Cl– DFDNB 1,5-Diuoro-2,4-dinitrobenzene MW 204.09 Spacer arm 3.0 Å N+ O– O N+ – O O F F 30 Protein interaction and crosslinking using mass spectrometry Chemical crosslinking in combination with mass spectrometry is a powerful method to determine protein–protein interactions. This method has been applied to recombinant and native protein complexes, and more recently to whole cell lysates and intact unicellular organisms, in efforts to identify protein–protein interactions on a global scale. Thermo Scientific™ MS-grade crosslinkers are available with different linker lengths and as isotopically labeled sets to help elucidate protein–protein interactions (Table 5). Simplification of the analysis of crosslinked proteins is essential for successful protein characterization. In addition to traditional crosslinkers, next-generation crosslinkers have been developed to address simplifying MS analysis through crosslinker enrichment and cleavable functionality. These high-quality reagents have been validated in protein–protein interaction studies using Thermo Scientific™ mass spectrometers that use different types of fragmentation (CID, HCD, ETD, and EtHCD) and levels of tandem mass spectrometry (MS2 and MS3 ), in order to improve identification of protein–protein interaction sites. Our MS-grade crosslinkers are high-quality reagents that are available in multiple packaging options and sizes. We offer extensive technical expertise and support for various applications, as well as validation of these products in workflows using Thermo Scientific™ mass spectrometers. Highlights • High quality—products manufactured in ISO 9001–certified facilities • Convenience—products available in Thermo Scientific™ No-Weigh™ packaging or in multiple pack sizes • More choices—available with different linker lengths, MS cleavability, and deuterium isotope labels • Technical support—extensive web resources and support to help ensure successful results Table 5. Overview of Thermo Scientific crosslinkers used for studying protein–protein interactions. Crosslinker DSS BS3 BS3 -d4 DSG Structure N O O O O O N O O O N O O O O O N O O O S O O S O O Na+O– O–Na+ N O O O O O N O O O S O O O S O D D D D Na+O– O–Na+ N O O O O O O N O O Full name Disuccinimidyl suberate Bis(sulfo-succinimidyl) suberate Bis(sulfo-succinimidyl) 2,2,7,7-suberate-d4 Disuccinimidyl glutarate Spacer arm (Å) 11.4 11.4 11.4 7.7 Water-soluble No Yes Yes No Isotopically labeled No No Yes No MS-cleavable No No No No Crosslinker BS2 G-d0 BS2 G-d4 DSSO DSBU Structure N O O O O S O O O N O O O S O O Na+O– O–Na+ N O O O O S O O O N O O O S O O D D D D Na+O– O–Na+ N O S O O O O N O O O O O O O O O O O O O N N N H N H C Full name Bis(sulfo-succinimidyl) glutarate Bis(sulfo-succinimidyl) 2,2,4,4-suberate-d4 Disuccinimidyl sulfoxide Disuccinimidyl dibutyric urea Spacer arm (Å) 7.7 7.7 10.1 12.5 Water-soluble Yes Yes No No Isotopically labeled No Yes No No MS-cleavable No No Yes Yes 31 MS-cleavable crosslinkers (DSSO and DSBU) Thermo Scientific™ DSSO (disuccinimidyl sulfoxide) and DSBU (disuccinimidyl dibutyric urea, also known as BuUrBu) are high-quality, MS-cleavable crosslinkers that contain an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a 7-atom and 11-atom spacer arm, respectively (Table 5, page 30). These products are offered in convenient single-use packaging (10 x 1 mg). Features of DSSO and DSBU include: • Amine-reactive NHS ester (at both ends) reacts rapidly with any molecule containing a primary amine • MS-cleavable by collision-induced dissociation (CID) • High-purity crystalline reagents for protein structure and interaction characterization • Membrane-permeant, allowing intracellular crosslinking • Water-insoluble (dissolve first in DMF or DMSO) The crosslinker facilitates analysis of protein structure and complex interactions using mass spectrometry. DSSO and DSBU have reactivity similar to that of DSS, but contain linkers that can be cleaved in the gas phase during tandem MS (MS2 ) using CID. The ability to cleave crosslinked peptides during MS2 enables MS3 acquisition methods, which facilitate peptide sequencing using traditional database search engines. The MS cleavage of DSSO and DSBU also generates diagnostic ion doublets during MS2 , which enables identification of crosslinked peptides from deadend modifications and searching using novel database search engines such as MeroX or XlinkX* (Figure 44). * Licensed from the Heck group, Utrecht University, The Netherlands. A. Spectra of DSSO-crosslinked peptide B. Spectra of DSBU-crosslinked peptide Figure 44. Spectra of BSA-crosslinked peptides identified by MS2 /MS3 method and XlinkX software. The peptides were crosslinked using (A) DSSO and (B) DSBU. XlinkX software uses unique fragment patterns of MS-cleavable crosslinkers to detect and filter crosslinked peptides for a database search. Ordering information Product Quantity Cat. No. DSSO (Disuccinimidyl Sulfoxide) 10 x 1 mg A33545 DSBU (Disuccinimidyl Dibutyric Urea) 10 x 1 mg A35459 32 Enrichable MS crosslinkers PhoX (DSPP) and TBDSPP Thermo Scientific™ PhoX (DSPP, disuccinimidyl phenyl phosphonic acid) and TBDSPP (tert-butyl disuccinimidyl phenyl phosphonate, tBu-PhoX) are amine-reactive, enrichable crosslinkers designed for mass spectrometry analysis. Both crosslinkers have amine-reactive N-hydroxysuccinimide (NHS) esters at the ends of a 7-atom spacer arm containing either a phosphonic acid group, PhoX (DSPP), or phosphonate ester (TBDSPP) for enrichment (Figure 45). These phospho groups are used for enrichment of crosslinked peptides using immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). Additionally, TBDSPP is cell-permeant for intracellular crosslinking applications. Figure 45. Chemical structure of enrichable crosslinkers PhoX (DSPP) and TBDSPP. O O P O O N N HO O OH O O O O PhoX (DSPP) O O P O O N N O O O O O O O TBDSPP Figure 46. Schematic of PhoX (DSPP) and TBDSPP crosslinking LC-MS workflows. Simple proteins and complexes are crosslinked using PhoX (DSPP) before digestion, phospho-enrichment, and LC-MS analysis. Proteins, organelles, or cells can be crosslinked using TBDSPP but require deprotection using TFA before phospho-enrichment and LC-MS analysis. P Crosslink PhoX (DSPP) P Crosslink TBDSPP Digest PhoX (DSPP) workflow tBu-PhoX workflow Digest Phospho-enrich Lyse Deprotect P Phospho-enrich P P P Analyze Analyze or P P P Features of PhoX (DSPP) and TBDSPP include: • Trifunctional crosslinker—reactive groups: NHS ester (both ends), phosphonic acid (PhoX (DSPP)) or phosphonate ester (TBDSPP) in the spacer for enrichment • High-purity crystalline reagents for protein structure and interaction characterization • Membrane-permeant version (TBDSPP) for intracellular crosslinking • Enrichable using Fe-NTA IMAC or TiO2 MOAC Of the two enrichable MS crosslinkers, PhoX (DSPP) is more water soluble than TBDSPP and is better for in vitro crosslinking of simple purified proteins and protein complexes. TBDSPP is better for intracelluar crosslinking, being more membrane-permeant. Athough both crosslinkers can be enriched using traditonal phosphopeptide methods, TBDSPP-crosslinked peptides must first be incubated with trifluoroacetic acid (TFA) to remove the tert-butyl protection groups (Figure 46). Chemical crosslinking in combination with MS is a powerful method to determine protein–protein interactions. This method has been applied to recombinant and native protein complexes and to whole cell lysates or intact unicellular organisms in efforts to identify protein–protein interactions on a global scale. Both traditional, noncleavable- and MS-cleavable crosslinkers can be used for identification of protein–protein interaction sites, but phospho-enrichable crosslinkers are advantageous because they can be used to enrich low-abundance crosslinked peptides and thereby improve MS identification rates (Figures 47 and 48). 33 Figure 47. BSA and ADH crosslinking using DSS and PhoX (DSPP). Purified proteins were crosslinked using a 20-fold molar excess and acetone-precipitated before digestion and LC-MS analysis. PhoX (DSPP) samples enriched using the High-Select Fe-NTA Magnetic Agarose Kit had >2-fold more crosslinks identified than DSS samples. Figure 48. Crosslinking of E. coli ribosomes. Ribosomes were crosslinked with a 40-fold molar excess of different crosslinkers for 1 hour at room temperature. Samples were reduced and alkylated before acetone precipitation to remove excess crosslinker. TBDSPP samples were deprotected for 30 minutes at 37°C with 2.5% TFA. All samples were digested overnight before C18 cleanup and LC-MS analysis. Ordering information Product Quantity Cat. No. DSPP (Disuccinimidyl Phenyl Phosphonic Acid, PhoX) 50 mg A52286 TBDSPP (tert-Butyl Disuccinimidyl Phenyl Phosphonate, tBu-PhoX) 50 mg A52287 BSA ADH BSA ADH BSA ADH DSS Crosslinker and protein 0 20 40 60 80 100 120 Number of crosslink identifications PhoX (DSPP) PhoX (DSPP) enriched 0 20 40 60 80 100 120 DSSO BS³ DSS DSG TBDSPP PhoX (DSPP) TBDSPP enriched PhoX (DSPP) enriched Number of crosslink identifications Crosslinker Learn more at thermofisher.com/ms-crosslinking 34 In vivo crosslinking Crosslinkers are used for identification of near-neighbor protein relationships and ligand–receptor interactions. Crosslinking stabilizes transient endogenous protein–protein complexes that may not survive traditional biochemical techniques such as immunoprecipitation. The most basic cellular crosslinker is formaldehyde (Thermo Scientific™ Pierce™ 16% Formaldehyde (w/v), Methanol-free, Cat. No. 28906), which is commonly used to stabilize chromatin interactions for chromatin immunoprecipitation (ChIP) assays (Thermo Scientific™ Pierce™ Agarose ChIP Kit, Cat. No. 26156). The crosslinkers chosen for these applications are usually longer than those used for subunit crosslinking. Homobifunctional amine-reactive NHS esters or imidates and heterobifunctional amine-reactive, photoactivatable phenyl azides are the most commonly used crosslinkers for these applications. Occasionally, a sulfhydryl- and aminereactive crosslinker such as Thermo Scientific™ Sulfo-SMCC (Cat. No. 22322) may be used if one of the two proteins or molecules is known to contain sulfhydryls. Both cleavable or noncleavable crosslinkers can be used. Because the distances between two molecules are not always known, the optimal length of the spacer arm of the crosslinker may be determined using a panel of similar crosslinkers with different lengths. Thermo Scientific™ DSS (Cat. No. 21555) and its cleavable analog DSP (Cat. No. 22585) are among the shorter crosslinkers used for protein–protein interactions. In contrast to crosslinkers that are introduced to cells exogenously, methods exist to incorporate crosslinkers into the proteome of a cell. This can be accomplished with photoreactive crosslinkers such as Thermo Scientific™ L-Photo-Leucine (Cat. No. 22610) and L-Photo-Methionine (Cat. No. 22615). These amino acid analogs are fed to cells during cell growth and are activated with UV light (Figure 49). In the experiment below, photoreactive amino acids were compared to formaldehyde treatment for identifying endogenous protein complexes (Figure 50). Figure 49. In vivo crosslinking with photoreactive amino acids. Learn more at thermofisher.com/in-vivo-crosslinking 1 2 3 1 2 3 1 2 3 1 2 3 α-HSP90 α-Stat3 α-Ku70 α-Ku86 Figure 50. Photoreactive amino acid crosslinking and formaldehyde crosslinking are complementary techniques for protein interaction analysis. HeLa cells were mock treated (lane 1), treated with 1% formaldehyde for 10 minutes (lane 2), or treated with Photo-Methionine and Photo-Leucine followed by UV treatment (lane 3). Cells were lysed, and 10 µg of each was analyzed by SDS-PAGE and western blotting with antibodies against HSP90, Stat3, Ku70, and Ku86. Lower panels are β-actin (upper band) and GAPDH (lower band), which were blotted as loading controls. Crosslinked Crosslinked Monomer – + UV Add Photo-Met and Photo-Leu dissolved in limiting media to cells. Incubate 24 hours at 37°C, 5% CO2 . Expose cells to UV light (365 nm bulb). Analyze by western blot using specic antibody. UV Harvest cells, lyse. Run SDS-PAGE. OH O H N N OH O S H H2 N H2 N L-Photo-Methionine L-Methionine H2 N OH O N N H OH O H H2 N L-Photo-Leucine L-Leucine 35 Learn more at thermofisher.com/metabolic-labeling Metabolic labeling Chemoselective ligations use unique chemical functional groups for specific conjugation. Examples of this chemistry include hydrazide–aldehyde condensation, click chemistry (azide–alkyne), and Staudinger ligation (azide–phosphine). Click chemistry is the detection method of choice for samples that would be compromised by direct labeling or antibody-based secondary detection techniques. The click label is small enough to penetrate complex samples easily, and the selectivity and stability of the click reaction provides high sensitivity and low background signal. This gentle sample treatment together with the biocompatible Invitrogen™ Click-iT™ Plus reaction means that detection can be multiplexed with expressed proteins such as GFP, protein labels such as R-PE, and a wide range of organic fluorophores. The Staudinger ligation has the best utility for live-cell labeling and mass spectrometry (MS) applications. The Staudinger reaction occurs between a phosphine and an azide to produce an aza-ylide that is trapped to form a stable covalent bond. Because phosphines and azides are absent in biological systems, there is minimal background labeling of cells or lysates. Unlike click chemistry, Staudinger ligation requires no accessory reagents such as copper. Metabolic labeling involves incorporation of a chemoselective crosslinker into the proteome of living cells. This facilitates protein isolation or fluorescent labeling. One such example is the metabolic labeling of glycoproteins using azido sugars. Azido groups react with phosphines to create a stable amide bond following the Staudinger reaction. Once incorporated, azido sugars can be labeled with biotin (Figure 51) or DyLight fluorophores (Figure 52). In this experiment, U2OS cells or HK-2 cells were fed azido sugars, fixed, and stained with Thermo Scientific™ DyLight™ 550-Phosphine (Cat. No. 88910) or DyLight™ 650-Phosphine (Cat. No. 88911). O AcO AcO NH OAc OAc N3 O GlcNAz N-Azidoacetylglucosamine O AcO NH OAc OAc AcO N3 O N-Azidoacetylgalactosamine GalNAz O O AcO AcO HN OAc AcO N3 N-Azidoacetylmannosamine ManNAz O O HO OH H N N3 N O HO OH NH HN S O O H N O H N H H O PPh2 OCH3 O O 3 3 O O HO OH H N O HN CO2 – CO2 – O HO OH NH NH HN S O O H N O H N H H O Ph2P O O New amide bond Azido sugar Biotin-PEG3 -phosphine Extracellular Intracellular Figure 51. Biotin labeling of azido sugars. U2OS: azido-galactosamine A B HK-2: azido-mannosamine Figure 52. In vivo detection of metabolically incorporated azido sugars using DyLight 550- and 650-Phosphine labeling reagents. (A) U2OS cells were incubated with 40 µM azido-acetylgalactosamine in cell culture medium for 72 hours, and the live cells were incubated with 100 µM of DyLight 550-Phosphine. The cells were then washed, fixed with 4% paraformaldehyde, and counterstained with Hoechst™ 33342 stain (green: DyLight 550–labeled azido-galactosamine, blue: Hoechst 33342–labeled nuclei). (B) HK-2 cells were incubated with 40 µM azidoacetylmannosamine in cell culture medium for 72 hours, and the live cells were incubated with 100 µM of DyLight 650-Phosphine. The cells were then washed, fixed with 4% paraformaldehyde, and counterstained with Hoechst 33342 stain (cyan: DyLight 650–labeled azido-mannosamine, red: Hoechst 33342–labeled nuclei). Cell surface crosslinking and biotinylation Crosslinkers are often used to identify surface receptors or their ligands. Membrane-impermeant crosslinkers ensure cell surface–specific crosslinking. Water-insoluble crosslinkers, when used in controlled amounts of reagent and reaction times, can reduce membrane penetration and reaction with inner-membrane proteins. The sulfonyl groups attached to the succinimidyl rings of NHS esters result in a crosslinker that is water-soluble, membraneimpermeant, and nonreactive with inner-membrane proteins. Therefore, reaction time and quantity of crosslinker are less critical when using sulfo-NHS esters. Homobifunctional sulfo-NHS esters, heterobifunctional sulfo-NHS esters, and photoreactive phenyl azides are good choices for crosslinking proteins on the cell surface (Figure 53). Determination of whether a particular protein is located on the surface or the integral part of the membrane can be achieved by performing a conjugation reaction of a cell membrane preparation to a known protein or radioactive label using a water-soluble or water-insoluble crosslinker. Upon conjugation the cells may be washed, solubilized, and characterized by SDS-PAGE to determine whether the protein of interest was conjugated. Integral membrane proteins will form a conjugate in the presence of a water-insoluble crosslinker, but not in the presence of water-soluble crosslinkers. Surface membrane proteins can conjugate in the presence of water-soluble and water-insoluble crosslinkers. The Thermo Scientific™ Pierce™ Cell Surface Protein Isolation Kit (Cat. No. 89881) is a complete set of reagents that utilizes a cell-impermeant, cleavable biotinylation reagent (Sulfo-NHS-SS-Biotin) for the selective biotinylation and subsequent purification of mammalian cell surface proteins to the exclusion of intracellular proteins. The labeled surface proteins are affinity-purified using Thermo Scientific™ NeutrAvidin™ agarose resin. Cell membrane structural studies Cell membrane structural studies require reagents of varying hydrophobicity to determine the location and the environment within a cell’s lipid bilayer. Fluorescent tags are used to locate proteins, lipids, or other molecules inside and outside the membrane. Various crosslinkers, with differing spacer arm lengths, can be used to crosslink proteins to associated molecules within the membrane to determine the distance between molecules. Successful crosslinking with shorter crosslinkers is a strong indication that two molecules are interacting in some manner. Failure to obtain crosslinking with a panel of shorter crosslinkers, while obtaining conjugation with the use of longer reagents, generally indicates that the molecules are located in the same part of the membrane, but are not interacting. Homobifunctional NHS esters, imidates, or heterobifunctional NHS ester/photoactivatable phenyl azides are commonly used for these procedures. Although imidoester crosslinkers (imidates) are water-soluble, they are still able to penetrate membranes. Sulfhydryl-reactive crosslinkers may be useful for targeting molecules with cysteines to other molecules within the membrane. Thermo Scientific™ EDC (Cat. No. 22980, 22981) and other water-soluble and -insoluble coupling reagent pairs are used to study membranes and cellular structure, protein subunit structure and arrangement, enzyme–substrate interactions, and cell-surface and membrane receptors (Figure 54). The hydrophilic character of EDC can result in much different crosslinking patterns in membrane and subunit studies than hydrophobic carbodiimides. Often it is best to attempt crosslinking with a water-soluble and water-insoluble carbodiimide to obtain a complete picture of the spatial arrangements or protein–protein interactions involved. O S S H N O NH HN S O O N O O S O O Na+ O– Figure 53. Sulfo-NHS-SS-biotin is used for the biotinylation and isolation of cell surface proteins. Figure 54. EDC can be used in coupling studies to study cell membrane structure. N N C NH Cl + – 36 Special packaging to meet specific bioconjugation needs Introduction 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. 38 No-Weigh packaging format for bioconjugation reagents Convenient, ready-to-use vials for single-use applications With the convenient Thermo Scientific™ No-Weigh™ packaging format, a ready-to-use solution can be made quickly and conveniently. This unique packaging format helps 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. 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 The No-Weigh packaging format is available in convenient, easy-to-handle screw cap vials for our most widely used protein modification reagents, including reducing agents, crosslinkers, and PEG- and biotin-labeling products (Table 6). Table 6. No-Weigh reagents. Reagent type No-Weigh reagents Amine-to-azide TFP ester-PEG4-DBCO Carboxyl-to-amine crosslinkers EDC DSG Sulfo-NHS Amine-to-amine crosslinkers DSP BS3 DSS DSSO DSBU BS(PEG)5 Amine-to-sulfhydryl crosslinkers SMCC Sulfo-SMCC SM(PEG)2 SM(PEG)12 Photoreactive Sulfo-SANPAH Modification reagents Iodoacetamide Reducing agents TCEP-HCl DTT Sulfhydryl-to-azide Maleimide-PEG4-DBCO 39 Premium-grade protein bioconjugation reagents Higher quality and assurance of performance from a trusted supplier Thermo Scientific™ Pierce™ Premium-Grade Reagents are the reagents of choice for applications where product integrity and risk minimization are critical (Table 7). 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. Pierce Premium-Grade Reagents are manufactured to the highest possible specifications to enable data integrity and offer robust consistency. The consistency of each lot of reagent is assessed using thorough testing procedures (Table 8). 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 • Change management—change control notification (CCN) service • Consistent manufacturing—batch-specific manufacturing documentation review Table 7. Pierce Premium-Grade Reagents. Reagent type Pierce Premium-Grade Reagent Carboxyl-to-amine crosslinkers EDC Sulfo-NHS Amine-to-amine crosslinkers BS3 DSP Amine-to-sulfhydryl crosslinkers Sulfo-SMCC SPDP Biotinylation reagents Sulfo-NHS-LC-biotin Sulfo-NHS-SS-biotin Reducing agents TCEP-HCl Table 8. Testing for Pierce Premium-Grade Reagents. Specification Procedure Purity Quantitative NMR using an internal standard Visual Color assessment Solubility Example: sample dissolves at a specified concentration in a given solvent to yield a clear, colorless solution Identity Infrared (IR) spectroscopy Mass identity Mass spectrometry Water content Karl Fischer titration Trace metals Inductively coupled plasma mass spectrometry (ICP-MS) Elemental analysis Reported values for C, H, N, O, and S based on combustion analysis Residual solvent analysis* Headspace gas chromatography * Performed upon request at an additional cost. 40 Bioconjugation resources Crosslinker Selection Tool The Crosslinker Selection Tool provides quick access to customized lists of Thermo Scientific™ Pierce™ crosslinkers that meet specific criteria, including target functional group, solubility, and cell membrane and the ability to permeate the cell membrane. Use the simple drop-down boxes to easily select the optimal crosslinking reagent for your application. Learn more at thermofisher.com/crosslinking-tool Biotinylation Reagent Selection Tool The Biotinylation Reagent Selection 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 drop-down boxes to easily select the optimal biotinylation reagent for your application. Learn more at thermofisher.com/biotinylation-tool 41 Fluorescence SpectraViewer Tool The SpectraViewer is a tool to help you plan your experiments and analyses, by assisting you in choosing the best fluorophore based on your application, light sources, and filters. Easily find the right fluorophore to label your protein, antibody, or nucleic acid over a broad range of applications and techniques. Learn more at thermofisher.com/spectraviewer Labeling kit selection guide The labeling kit selection guide provides quick access to customized lists of antibody and protein labeling kits for fluorophore, biotin, or HRP labeling. Easily find the right labeling kit based on your application, light sources, sample prep needs, and scale. Learn more at thermofisher.com/AbLabelingGuide 42 Glossary of crosslinking terms Acylation: Reaction that introduces an acyl group (–COR) into a compound. Aryl azide: Compound containing a photoreactive functional group (e.g., phenyl azide) that reacts nonspecifically with target molecules. Carbodiimide: Reagent that catalyzes the formation of an amide linkage between a carboxyl (–COOH) group and a primary amine (–NH2 ) or a hydrazide (–NHNH2 ). These reagents do not result in the formation of a cross-bridge and have been termed zerolength crosslinkers. Crosslinker: Reagent that will react with functional groups on two or more molecules to form a covalent linkage between the molecules. Conjugation reagent: Crosslinker or other reagent for covalently linking two molecules. Diazirine crosslinker: Succinimidyl-ester diazirine (SDA) crosslinkers combine amine-reactive chemistry with an efficient diazirine-based photochemistry for photocrosslinking to nearly any other functional group. The photoactivation of diazirine with long-wave UV light (330–370 nm) creates carbene intermediates. These intermediates can form covalent bonds via addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths. Disulfide bonds: Oxidized form of sulfhydryls (–S–S–); formed in proteins through –SH groups from two cysteine molecules. These bonds often link polypeptide chains together within the protein and contribute to a protein’s tertiary structure. α-Haloacyl: Functional group (e.g., iodoacetyl) that targets nucleophiles, especially thiols. α-Haloacyl compounds have a halogen atom such as iodine, chlorine, or bromine attached to an acyl group on the molecule. These alkylating reagents degrade when exposed to direct light or reducing agents, resulting in loss of the halogen and the appearance of a characteristic color. Hapten: Molecule recognized by antibodies but unable to elicit an immune response unless attached to a carrier protein. Haptens are usually, but not always, small (<5 kDa) molecules. Homobifunctional crosslinker: Reagent with two identical reactive groups used to link two molecules or moieties. Heterobifunctional crosslinker: Reagent with two different reactive groups used to link two molecules or moieties. Hydrophilic: Substances that readily dissolve in water. Hydrophobic: Substances with limited solubility in water. N-hydroxysuccinimidyl (NHS) ester: Acylating reagent commonly used for crosslinking or modifying proteins. These esters are specific for primary (–NH2 ) amines between pH 7 and 9, but are generally the most effective at neutral pH. They are subject to hydrolysis, with half-lives approximating 1–2 hours at room temperature at neutral pH. Imidate crosslinker: Primary amine-reactive functional group that forms an amidine bond. The ε-amine in lysine and N-terminal amines are the targets in proteins. Imidates react with amines in alkaline conditions (pH 7.5–10) and hydrolyze quickly, with half-lives typically around 10–15 minutes at room temperature and pH 7–9. At pH >11, the amidine bond is unstable, and crosslinking can be reversed. The amidine bond is protonated at physiological pH; therefore, it carries a positive charge. Imidoester: Amine-reactive functional group of an imidate crosslinker. Immunogen: Substance capable of eliciting an immune response. Integral membrane protein: Protein that extends through the cell membrane and is stabilized by hydrophobic interactions within the lipid bilayer of the membrane. Ligand: Molecule that binds specifically to another molecule. For example, a protein that binds to a receptor. Moiety: Indefinite part of a sample or molecule. Monomer: Single unit of a molecule. NHS: Abbreviation for N-hydroxysuccinimide. Nitrene: Triple-bonded nitrogen–nitrogen reactive group formed after exposure of an azido group to UV light. Its reactivity is nonspecific and short lived. Nonselective crosslinking: Crosslinking using reactive groups such as nitrenes or aryl azides, which react so quickly and broadly that specific groups are not easily and efficiently targeted. Yields are generally low, with many different crosslinked products formed. Nonspecific crosslinking: Another term for nonselective crosslinking. Oligomer: Molecule composed of several monomers. 43 Photoreactive: Reactive upon excitation with light at a particular range of wavelengths. Polymer: Molecule composed of many repeating monomers. Pyridyl disulfide: Aromatic moiety with a disulfide attached to one of the carbons adjacent to the nitrogen in a pyridine ring. Pyridine 2-thione is released when this moiety reacts with a sulfhydryl (–SH)-containing compound. Spacer arm: Part of a crosslinker that is incorporated between two crosslinked molecules and serves as a bridge between the molecules. Substrate: Substance upon which an enzyme acts. Sulfhydryl: –SH group, present on cysteine residues in proteins. Thiols: Also known as mercaptans, thiolanes, sulfhydryls, or –SH groups, these are good nucleophiles that may be targeted for crosslinking. Ultraviolet (UV): Electromagnetic radiation of wavelengths between 10 and 390 nm. 44 Ordering information Homobifunctional crosslinkers Product MW Spacer arm (Å) Structure Quantity Cat. No. Amine-to-amine reactive (NHS ester) DSG (disuccinimidyl glutarate) 326.26 7.7 N O O O O O O N O O 50 mg 20593 Pierce DSG, No-Weigh Format 10 x 1 mg A35392 DSS (disuccinimidyl suberate) 368.35 11.4 N O O O O O N O O O 50 mg 21655 1 g 21555 Pierce DSS, No-Weigh Format 10 x 2 mg A39267 BS3 (bis(sulfosuccinimidyl)suberate) 572.43 11.4 N O O O O O N O O O S O O S O O Na+O– O–Na+ 50 mg 21580 1 g 21586 Pierce Premium-Grade BS3 100 mg PG82083 1 g PG82084 Pierce BS3 , No-Weigh Format 10 x 2 mg A39266 BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate) 532.50 21.7 N O O O O O O O O O N O O O O 100 mg 21581 Pierce BS(PEG)5 , No-Weigh Format 10 x 1 mg A35396 BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate) 708.71 35.8 N O O O O O O O O O N O O O O O O O O 100 mg 21582 DSP (dithiobis(succinimidyl propionate)), Lomant’s Reagent 404.42 12.0 S O S N O O O O N O O O 1 g 22585 50 mg 22586 Pierce Premium-Grade DSP 1 g PG82081 10 g PG82082 Pierce DSP, No-Weigh Format 10 x 1 mg A35393 DTSSP (3,3´-dithiobis(sulfosuccinimidyl propionate)) 608.51 12.0 S N S O O O O O N O O O S O O S O O Na+O– O–Na+ 50 mg 21578 DST (disuccinimidyl tartrate) 344.24 6.4 O O N O O O N O O OH O OH 50 mg 20589 EGS (ethylene glycol bis(succinimidyl succinate)) 456.36 16.1 O O N O O O N O O O O O O O 1 g 21565 Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)) 660.45 16.1 O O O N O O O O O N O O O S O O O S O O O–Na+ Na+O– 50 mg 21566 TSAT (tris-(succinimidyl) aminotriacetate) 482.36 4.2 N O O O O O N O O O N O O N O O 50 mg 33063 45 Product MW Spacer arm (Å) Structure Quantity Cat. No. Amine-to-amine reactive (NHS ester), deuterated or MS-cleavable BS2 G-d0 (bis(sulfosuccinimidyl) glutarate-d0) 530.35 7.7 N O O O O S O O O N O O O S O O Na+O– O–Na+ 10 mg 21610 BS2 G-d4 (bis(sulfosuccinimidyl) 2,2,4,4-glutarate-d4 ) 534.38 7.7 N O O O O S O O O N O O O S O O D D D D Na+O– O–Na+ 10 mg 21615 BS3 -d0 (bis(sulfosuccinimidyl) suberate-d0) 572.43 11.4 N O O O O O N O O O S O O S O O Na+O– O–Na+ 10 mg 21590 BS3 -d4 (bis(sulfosuccinimidyl) 2,2,7,7-suberate-d4 ) 576.45 11.4 N O O O O O N O O O S O O O S O D D D D Na+O– O–Na+ 10 mg 21595 DSSO (disuccinimidyl sulfoxide) 388.35 10.1 N O S O O O O N O O O O 10 x 1 mg A33545 DSBU (disuccinimidyl dibutyric urea) 426.38 12.5 O O O O O O O O O N N N H N H C 10 x 1 mg A35459 Amine-to-amine reactive (imidoester or difluoro) DMP (dimethyl pimelimidate) 259.17 9.2 O O NH2 +Cl– NH2 +Cl– 50 mg 21666 1 g 21667 DMS (dimethyl suberimidate) 273.20 11.0 O O NH2 +Cl– NH2 +Cl– 1 g 20700 DTBP (Wang and Richard’s Reagent) 309.28 11.9 S S O O NH2 +Cl– NH2 +Cl– 1 g 20665 DFDNB (1,5-difluoro-2,4-dinitrobenzene) 204.09 3.0 N+ O– O N+ –O O F F 50 mg 21525 Learn more at thermofisher.com/proteincrosslinking 46 Product MW Spacer arm (Å) Structure Quantity Cat. No. Sulfhydryl-to-sulfhydryl reactive (maleimide) BMOE (bismaleimidoethane) 220.18 8.0 N O O N O O 50 mg 22323 BMB (1,4-bismaleimidobutane) 248.23 10.9 N O O N O O 50 mg 22331 BMH (bismaleimidohexane) 276.29 16.1 N O O N O O 50 mg 22330 BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) 308.29 14.7 O O N O O N O O 50 mg 22336 BM(PEG)3 (1,11-bismaleimido-triethyleneglycol) 352.34 17.8 O O O N O O N O O 50 mg 22337 DTME (dithiobismaleimidoethane) 312.37 13.3 S S N O O N O O 50 mg 22335 TMEA (tris(2-maleimidoethyl)amine) 386.36 10.3 N N O O N O O N O O 50 mg 33043 47 Product MW Spacer arm (Å) Structure Quantity Cat. No. Amine-to-sulfhydryl reactive (NHS-haloacetyl) SIA (succinimidyl iodoacetate) 283.02 1.5 N O O O I O 50 mg 22349 SBAP (succinimidyl 3-(bromoacetamido)propionate) 307.10 6.2 N O O O H N O O Br 50 mg 22339 SIAB (succinimidyl (4-iodoacetyl) aminobenzoate) 402.14 10.6 O O N H N O O O I 50 mg 22329 Sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate) 504.19 10.6 O O H N O I N O O S Na+O– O O 50 mg 22327 Amine-to-sulfhydryl reactive (NHS-maleimide) AMAS (N-α-maleimidoacetoxysuccinimide ester) 252.18 4.4 O O N N O O O O 50 mg 22295 BMPS (N-β-maleimidopropyloxysuccinimide ester) 266.21 5.9 N O O O O N O O 50 mg 22298 GMBS (N-γ-maleimidobutyryloxysuccinimide ester) 280.23 7.3 O O O O O O N N 50 mg 22309 Sulfo-GMBS (N-γ-maleimidobutyryloxysulfosuccinimide ester) 382.28 7.3 O O O N O O S Na+O– O O O N 50 mg 22324 MBS (m-maleimidobenzoylN-hydroxysuccinimide ester) 314.25 7.3 O O N O O N O O 50 mg 22311 Sulfo-MBS (m-maleimidobenzoylN-hydroxysulfosuccinimide ester) 416.30 7.3 O O N O O N O O S Na+O– O O 50 mg 22312 SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) 334.32 8.3 N O O O N O O O 50 mg 22360 Pierce SMCC, No-Weigh Format 10 x 1 mg A35394 Heterobifunctional crosslinkers 48 Product MW Spacer arm (Å) Structure Quantity Cat. No. Sulfo-SMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane1-carboxylate) 436.37 8.3 O O N O O N O O S Na+O– O O 50 mg 22322 1 g 22122 Pierce Premium-Grade Sulfo-SMCC 100 mg PG82085 1 g PG82086 Pierce Sulfo-SMCC, No-Weigh Format 10 x 2 mg A39268 Pierce EMCA (N-ε-maleimidocaproic acid) 211.21 9.4 N O O OH O 1 g 22306 EMCS(N-ε-maleimidocaproyloxysuccinimide ester) 308.29 9.4 N O O O O N O O 50 mg 22308 Sulfo-EMCS (N-ε-maleimidocaproyloxysulfosuccinimide ester) 410.33 9.4 N O O O O N O O S Na+O– O O 50 mg 22307 SMPB (succinimidyl 4-(p-maleimidophenyl)butyrate) 356.33 11.6 O N O O N O O O 50 mg 22416 Sulfo-SMPB (sulfosuccinimidyl 4-(N-maleimidophenyl)butyrate) 458.38 11.6 O N O O N O O O S Na+O– O O 50 mg 22317 SMPH (succinimidyl 6-((β-maleimidopropionamido)hexanoate)) 379.36 14.2 N H O N O O O O N O O 50 mg 22363 LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy- (6-amidocaproate)) 447.48 16.2 N H O N O O O O N O O 50 mg 22362 Sulfo-KMUS (N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester) 480.46 16.3 O O N O O N O O S Na+O– O O 50 mg 21111 SM(PEG)2 (PEGylated SMCC crosslinker) 425.39 17.6 H N O O O N O O O N O 2 O 100 mg 22102 1 g 22103 Pierce SM(PEG)2 , No-Weigh Format 10 x 1 mg A35397 SM(PEG)4 (PEGylated SMCC crosslinker) 513.5 24.6 H N O O O O O O N O O O N O O 100 mg 22104 1 g 22107 49 Product MW Spacer arm (Å) Structure Quantity Cat. No. SM(PEG)6(PEGylated, long-chain SMCC crosslinker) 601.60 32.5 H N O O O O O O O N N O O O O O O 100 mg 22105 SM(PEG)8 (PEGylated, long-chain SMCC crosslinker) 689.71 39.2 O O O O O O O N O O O O O H N O N O O 100 mg 22108 SM(PEG)12 (PEGylated, long-chain SMCC crosslinker) 865.92 53.4 O O O O O O O N O O O O O O O O O H N O N O O 100 mg 22112 1 g 22113 Pierce SM(PEG)12, No-Weigh Format 10 x 1 mg A35398 SM(PEG)24 (PEGylated, long-chain SMCC crosslinker) 1,394.55 95.2 H N O O O N O O O N O 24 O 100 mg 22114 Amine-to-sulfhydryl reactive (NHS-pyridyldithiol) SPDP (succinimidyl 3-(2-pyridyldithio) propionate) 312.37 6.8 S O S O N O N O 50 mg 21857 Pierce Premium-Grade SPDP 100 mg PG82087 1 g PG82088 LC-SPDP (succinimidyl 6-(3(2-pyridyldithio)propionamido) hexanoate) 425.52 15.7 O N H N O S S O O N O 50 mg 21651 Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3´-(2-pyridyldithio)propionamido) hexanoate) 527.57 15.7 O N H N O S S O O N O S O O Na+O– 50 mg 21650 SMPT (4-succinimidyloxycarbonylα-methyl-α(2-pyridyldithio)toluene) 388.46 20.0 O O N O S S O N 50 mg 21558 PEG4-SPDP (PEGylated, long-chain SPDP crosslinker) 559.17 25.7 O O O O O O H N S S N N O O O 100 mg 26128 PEG12-SPDP (PEGylated, long-chain SPDP crosslinker) 912.07 54.1 O O O O 12 N H S S N N O O 100 mg 26129 Carboxyl-to-amine reactive (carbodiimide plus NHS ester) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) 191.70 NA N N C NH Cl + – 10 mg 77149 5 g 22980 25 g 22981 Pierce Premium-Grade EDC 1 g PG82079 25 g PG82073 500 g PG82074 Pierce EDC, No-Weigh Format 10 x 1 mg A35391 NHS (N-hydroxysuccinimide) 115.09 NA O O N HO 25 mg 24500 50 Product MW Spacer arm (Å) Structure Quantity Cat. No. Sulfo-NHS (N-hydroxysulfosuccinimide) 217.13 NA N HO O O S O– O O 500 mg 24510 5 g 24525 Pierce Premium-Grade Sulfo-NHS 500 mg PG82071 10 g PG82072 Pierce Sulfo-NHS, No-Weigh Format 10 x 2 mg A39269 Sulfhydryl-to-carbohydrate or -carboxyl (maleimide, pyridyldithiol/hydrazide, or isocyanate) BMPH (N-β-maleimidopropionic acid hydrazide) 297.19 8.1 H N O N O O NH3 + CF3 –O O 50 mg 22297 EMCH (N-ε-maleimidocaproic acid hydrazide) 225.24 11.8 N O O O N H NH3 + CF3 –O O 50 mg 22106 MPBH (4-(4-N-maleimidophenyl) butyric acid hydrazide) 309.75 17.9 H N O N O O NH3 +Cl– 50 mg 22305 KMUH (N-κ-maleimidoundecanoic acid hydrazide) 295.38 19.0 O H N N O O NH3 + CF3 –O O 50 mg 22111 PDPH (3-(2-pyridyldithio)propionyl hydrazide) 229.32 9.2 H N NH2 S S O N 50 mg 22301 PMPI (p-maleimidophenyl isocyanate) 214.18 8.7 N O O N C O 50 mg 28100 Photoreactive (NHS ester and aryl azide, phenyl azide, diazirine, or psoralen) Sulfo-SANPAH (sulfosuccinimidyl 6-(4´-azido-2´-nitrophenylamino) hexanoate) 492.40 18.2 O O N H N+ –O O N N+ N– N O O S Na+O– O O 50 mg 22589 Pierce Sulfo-SANPAH, No-Weigh Format 10 x 1 mg A35395 SDA (NHS-Diazirine) (succinimidyl 4,4´-azipentanoate) 225.20 3.9 O O O O N N N 50 mg 26167 Sulfo-SDA (Sulfo-NHS-Diazirine) (sulfosuccinimidyl 4,4´-azipentanoate) 327.25 3.9 Na+O– O O O O O O N N N S 50 mg 26173 LC-SDA (NHS-LC-Diazirine) (succinimidyl 6-(4,4´-azipentanamido) hexanoate) 338.36 12.5 H N O O O N O O N N 50 mg 26168 Sulfo-LC-SDA (Sulfo-NHS-LCDiazirine) (sulfosuccinimidyl 6-(4,4´-azipentanamido)hexanoate) 440.40 12.5 Na+O– O O O O O O O H N N N N S 50 mg 26174 51 Product MW Spacer arm (Å) Structure Quantity Cat. No. SDAD (NHS-SS-Diazirine) (succinimidyl 2-((4,4´-azipentanamido) ethyl)-1,3´-dithiopropionate) 388.46 13.5 O H N O N N O O O N S S 50 mg 26169 Sulfo-SDAD (Sulfo-NHS-SSDiazirine) (sulfosuccinimidyl 2-((4,4´-azipentanamido)ethyl)- 1,3´-dithiopropionate) 490.51 13.5 O H N N N Na+O– O O O O O O S N S S 50 mg 26175 SPB (succinimidyl-(4-(psoralen8-yloxy))-butyrate) 385.32 8.6 O O O O N O O O O 50 mg 23013 Sulfo-SBED Biotin Label Transfer Reagent 879.97 19.1 13.7 9.1 HN S S S O HN O H N O N O O O HN NH O N O N+ NS O O Na+O13.7 Å 9.1 Å 19.1 Å 10 mg 33033 Pierce Sulfo-SBED Biotin Label Transfer Reagent, No-Weigh Format 10 x 1 mg A39260 Chemoselective ligation (NHS ester and azide-phosphine or -alkyne) NHS-Azide 198.14 2.5 N3 O O N O O 10 mg 88902 NHS-PEG4-Azide 388.37 18.9 N3 O O O O O N O O O 100 mg 26130 NHS-Phosphine 461.40 5.4 P O O O O N O O 10 mg 88900 Iodoacetamide Alkyne 223.01 7.8 O ICH2 C NHCH2 C CH 1 mg I10189 Click-iT AHA (L-azidohomoalanine) 258.16 NA O NCH2 CH2 NH2 • CF3 COOH N N CH C OH – + 5 mg C10102 Click-iT HPG (L-homopropargyl-glycine) 127.14 NA HC CCH2 CH2 CH C OH O NH2 5 mg C10186 52 Learn more at thermofisher.com/proteincrosslinking Product MW Spacer arm (Å) Structure Quantity Cat. No. Chemoselective ligation GlcNAz (N-azidoacetylglucosamine, tetraacylated) 430.37 NA O AcO AcO NH N3 O OAc OAc 5 mg 88903 GalNAz (N-azidoacetylgalactosamine, tetraacylated) 430.37 NA O AcO NH N3 O OAc OAc AcO 5 mg 88905 ManNAz (N-azidoacetylmannosamine, tetraacylated) 430.37 NA O AcO AcO OAc AcO HN N3 O 5 mg 88904 TFP Ester-PEG4-DBCO 714.7 17.9 N O O H N O O O F F F F 4 25 mg C20039 10 x 1 mg C20043 TFP Ester-PEG12-DBCO 1,067.12 46.3 N O O H N O O O F F F F 12 25 mg C20040 Maleimide-PEG4-DBCO 647.74 29.75 N O N H O O H N O N O 4 O 25 mg C20041 10 x 1 mg C20044 Photoreactive amino acids L-Photo-Leucine 143.15 0 H2 N OH O N H N 100 mg 22610 L-Photo-Methionine 157.17 0 OH O H N N H2 N 100 mg 22615 53 Product MW Spacer arm (Å) Structure Quantity Cat. No. Amine-reactive EZ-Link NHS-Biotin 341.38 13.5 N O O O O NH HN S O 100 mg 20217 EZ-Link NHS-Desthiobiotin 311.33 9.7 H N N H O O O N O O 50 mg 16129 EZ-Link Sulfo-NHS-Biotin 443.43 13.5 N O O O O S O O NH HN S O Na+ O– 50 mg 21217 EZ-Link Sulfo-NHS-Biotin, No-Weigh Format 10 x 1 mg A39256 EZ-Link NHS-LC-Biotin 454.54 22.4 O O H N O NH HN S O N O O 50 mg 21336 EZ-Link Sulfo-NHS-LC-Desthiobiotin, No-Weigh Format 526.54 17.3 H N N H N H O O O O N O O S Na+O– O O 5 x 1 mg A39265 EZ-Link Sulfo-NHS-LC-Biotin 556.59 22.4 O O H N O NH HN S O N O O O S O Na+ O– 100 mg 21335 Pierce Premium-Grade Sulfo-NHS-LC-Biotin 100 mg PG82075 1 g PG82076 EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh Format 10 x 1 mg A39257 EZ-Link NHS-LC-LC-Biotin 567.70 30.5 N O O O O N H O H N O NH HN S O 50 mg 21343 EZ-Link Sulfo-NHS-LC-LC-Biotin 669.75 30.5 N O O O S O O O N H O H N O NH HN S O Na+ O– 50 mg 21338 EZ-Link Sulfo NHS-LC-LC-Biotin, No-Weigh Format 10 x 1 mg A35358 EZ-Link NHS-SS-Biotin 504.65 24.3 O S S H N O NH HN S O O N O O 50 mg 21441 Biotin and desthiobiotin labeling reagents 54 Product MW Spacer arm (Å) Structure Quantity Cat. No. EZ-Link Sulfo-NHS-SS-Biotin 606.69 24.3 O S S H N O NH HN S O O N O O S O O Na+ O– 100 mg 21331 Pierce Premium-Grade Sulfo-NHS-SS-Biotin 100 mg PG82077 1 g PG82078 EZ-Link Sulfo-NHS-SS-Biotin, No-Weigh Format 10 x 1 mg A39258 EZ-Link NHS-PEG4-Biotin 588.67 29.0 O O O O H N HN NH S O O O O N O O 25 mg 21330 50 mg 21362 1 g 21363 EZ-Link NHS-PEG4-Biotin, No-Weigh Format 10 x 2 mg A39259 EZ-Link NHS-PEG12-Biotin 941.09 56.0 H N S HN NH O O O O O N O O [ ] 12 25 mg 21312 500 mg 21313 EZ-Link NHS-PEG12-Biotin, No-Weigh Format 10 x 1 mg A35389 Sulfhydryl-reactive EZ-Link BMCC-Biotin 533.68 32.6 N H H N O NH HN S O O N O O 50 mg 21900 EZ-Link HPDP-Biotin 539.78 29.2 H N NH HN S O N H O S N S O 50 mg 21341 EZ-Link HPDP-Biotin, No-Weigh Format 10 x 1 mg A35390 EZ-Link Iodoacetyl-PEG2 -Biotin 542.43 24.7 HN O NH S O N O H H N O I O 50 mg 21334 EZ-Link Maleimide-PEG2 -Biotin 525.62 29.1 N H O O NH HN S O O H N O N O O 50 mg 21901BID EZ-Link Maleimide-PEG2 -Biotin, No-Weigh Format 10 x 2 mg A39261 Learn more at thermofisher.com/biotinlabeling 55 Modification reagents for reduction and denaturation of proteins Product MW Spacer arm (Å) Reactive group Structure Quantity Cat. No. Disulfide bond reduction Pierce 2-Mercaptoethanol/ (β-mercaptoethanol) 78.13 NA Thiol HS OH 10 x 1 mL 35602 Pierce Mercaptoethylamine-HCl (2-mercaptoethylamine-HCl (2-MEA)) 78.13 NA Thiol HS NH3 + Cl– 6 x 6 mg 20408 Pierce Cysteine-HCl 175.63 NA Thiol NH3 + Cl– HS O HO H2 O 5 g 44889 Pierce DTT, Cleland’s Reagent/Dithiothreitol 154.25 NA Thiol SH HS OH OH 5 g 20290 Pierce DTT, No-Weigh Format 48 x 7.7 mg A39255 Bond-Breaker TCEP Solution, Neutral pH 286.25 NA Phosphine HO P O O OH OH O 5 mL 77720 Pierce TCEP-HCl 286.65 NA Phosphine O HO O OH O OH P 1 g 20491 10 g 20490 Pierce Premium-Grade TCEP-HCl 1 g PG82080 10 g PG82089 100 g PG82090 Schiff base reduction to alkylamine linkage AminoLink Reductant (sodium cyanoborohydride) 62.84 NA Cyanoborohydride N BH3 – Na+ 2 x 1 g 44892 Protein denaturants and chaotropes Guanidine-HCl 95.53 NA NA H2 N C NH2 ·HCl NH 500 g 24110 Guanidine-HCl (8 M solution) 200 mL 24115 Urea 60.06 NA NA H2 N NH2 O 1 kg 29700 Learn more at thermofisher.com/proteinmodification 56 Modification reagents for proteins and peptides Product MW Spacer arm (Å) Reactive group Structure Quantity Cat. No. Irreversibly blocks primary amines Pierce Sulfo-NHS-Acetate (sulfo-N-hydroxysulfosuccinimide acetate) 259.17 NA NHS ester O N O O O S Na +O– O O 100 mg 26777 Reversibly blocks primary amines Pierce Citraconic Anhydride (2-methylmaleic anhydride) 112.08 NA NA O O O H3 C 100 g 20907 Modifies primary amines to contain a protected sulfhydryl group Pierce SATP (N-succinimidyl S-acetylthio-propionate) 245.25 4.1 NHS ester N O O O S O O 50 mg 26100 Pierce SAT(PEG)4 (N-succinimidyl S-acetyl(thiotetraethylene glycol) 421.46 18.3 NHS ester/ acetylated sulfhydryl (protected) N O O O O O O S O O O 100 mg 26099 Pierce SATA (N-succinimidyl S-acetylthioacetate) 231.23 2.8 NHS ester N O O O O S O 50 mg 26102 Modifies primary amines to contain a free sulfhydryl group Traut’s Reagent (2-iminothiolane) 137.63 8.1 Iminothiolane S NH2 + Cl – 500 mg 26101 Adds amine or carboxylic acid functional group to protein or surface Pierce AEDP (3-((2-aminoethyl)dithio) propionic acid-HCl) 217.74 NA Amine/ carboxylic acid Cl- H3 +N S S OH O 50 mg 22101 Irreversibly blocks sulfhydryl groups Pierce NEM (N-ethylmaleimide) 125.13 NA Maleimide CH3 O O N 25 g 23030 Reversibly blocks sulfhydryl groups Pierce MMTS (methyl methanethiosulfonate) 126.20 NA NA H3C S O S CH3 O 200 mg 23011 57 Product MW Spacer arm (Å) Reactive group Structure Quantity Cat. No. Adds primary amine to glass and silica surfaces through silylation Pierce APTS (3-aminopropyltriethoxysilane) 221.37 NA NA O Si NH2 O O 100 g 80370 Oxidizes carbohydrates for reductive amination Pierce Sodium Meta-Periodate 213.89 NA Periodate O O I O– O Na+ 25 g 20504 Alkylates reduced cysteines Pierce Iodoacetic Acid 185.95 NA Iodoacetyl OH O I 500 mg 35603 Pierce Iodoacetamide, No-Weigh Format 184.96 NA Iodoacetyl NH2 O I 30 x 9.3 mg A39271 Pierce Chloroacetamide, No-Weigh Format 93.51 NA Chloroacetyl NH2 O Cl 10 x 2 mg A39270 Deprotects SATA-modified molecules Pierce Hydroxylamine-HCl 69.49 NA NA Cl H3 N+ OH – 25 g 26103 58 PEGylation (PEG labeling) reagents for proteins Product MW Spacer arm (Å) Reactive group Structure Quantity Cat. No. Amine-reactive linear PEGylation of protein or surface, terminating with a methyl group MS(PEG)4 (methyl-PEG4-NHS ester) 333.33 16.4 NHS ester O O O O O N O O O CH3 100 mg 22341 1 g 22342 MS(PEG)8 (methyl-PEG8-NHS ester) 509.40 30.8 NHS ester O O O N O O CH3 8 100 mg 22509 MS(PEG)12 (methyl-PEG12-NHS ester) 685.71 44.9 NHS ester O O O N O O CH3 12 100 mg 22685 1 g 22686 MS(PEG)24 (methyl-PEG24-NHS ester) 1,214.39 88.2 NHS ester O O O N O O CH3 24 100 mg 22687 Amine-reactive branched PEGylation of a protein or surface, terminating with a methyl group TMS(PEG)12 ((methyl-PEG12) 3-PEG4-NHS ester) 2,420.80 52.0 NHS ester H N O O O O O O O O O O O O CH3 O O O O O O O O O O O O CH3 O O N H O O H N O O O O O O O O O O O O O O CH3 N O H O O O O O NH 46.2 Å O O N O O 25.5 Å 52.0 Å 1 g 22424 Sulfhydryl-reactive branched PEGylation of a protein or surface, terminating with a methyl group MM(PEG)12 (methyl-PEG12-maleimide) 710.81 51.9 Maleimide O H N CH3 O N O O 12 100 mg 22711 MM(PEG)24 (methyl-PEG24-maleimide) 1,239.44 95.3 Maleimide O H N CH3 O N O O 24 100 mg 22713 PEGylation of a protein or surface, terminating with a carboxylic acid or primary amine CA(PEG)4 (carboxyl-(4-ethyleneglycol) ethylamine) 265.30 18.1 Amine/ carboxylic acid O O O O O HO NH2 100 mg 26120 1 g 26121 CA(PEG)8 (carboxyl-(8-ethyleneglycol) ethylamine) 441.51 33.6 Amine/ carboxylic acid [ ] 8 O HO O NH2 100 mg 26122 1 g 26123 CA(PEG)12 (carboxyl-(12-ethyleneglycol) ethylamine) 617.72 46.8 Amine/ carboxylic acid [ ] 12 O HO O NH2 100 mg 26124 1 g 26125 CA(PEG)24 (carboxyl-(24-ethyleneglycol) ethylamine) 1,146.35 89.8 Amine/ carboxylic acid [ ] 24 O HO O NH2 100 mg 26126 1 g 26127 Learn more at thermofisher.com/pegylation 59 Product MW Spacer arm (Å) Reactive group Structure Quantity Cat. No. PEGylation of a gold, silver, or metal surface, terminating with a carboxylic acid or methyl group CL(PEG)12 Carboxy-PEG-Lipoamide Compound 806.03 55.5 Carboxylic acid/ bidentate thiol H N O O HO O S S 12 100 mg 26135 CT(PEG)12 Carboxy-PEG-Thiol Compound 634.77 47.8 Carboxylic acid/ thiol SH O O O O O O O O O O O HO O O 100 mg 26133 ML(PEG)4 Methyl-PEG-Thiol Compound 395.58 23.6 Bidentate thiol H N O O O O H3 C O S S 100 mg 26134 PEGylation of a protein or inert material surface, terminating with a methyl group MT(PEG)4 Methyl-PEG-Thiol Compound 224.32 15.8 Thiol H3 C O O O O SH 100 mg 26132 PEGylation of a protein, oxidized carbohydrate, or surface, terminating with a methyl group MA(PEG)4 (methyl-(4-ethyleneglycol) ethylamine) 207.27 15.5 Amine O O O H2 N O CH3 100 mg 26110 1 g 26111 MA(PEG)8 (methyl-(8-ethyleneglycol) ethylamine) 383.48 29.7 Amine O H2 N CH3 8 100 mg 26112 1 g 26113 MA(PEG)12 (methyl-(12-ethyleneglycol) ethylamine) 559.69 43.9 Amine O H2 N CH3 12 100 mg 26114 1 g 26115 MA(PEG)24 (methyl-(24-ethyleneglycol) ethylamine) 1,088.32 86.1 Amine O H2 N CH3 24 100 mg 26116 1 g 26117 60 Product Emission/excitation (nm) Emission color Quantity Cat. No. Amine-reactive Alexa Fluor 350 NHS Ester (Succinimidyl Ester) 346/442 Blue 5 mg A10168 Alexa Fluor 350 Antibody Labeling Kit, 5 x 100 µg of antibody 346/442 Blue 1 kit A20180 Alexa Fluor 405 NHS Ester (Succinimidyl Ester) 402/421 Blue 1 mg A30000 5 mg A30100 Pacific Blue Succinimidyl Ester 385/445 Blue 5 mg P10163 BODIPY 493/503 NHS Ester (Succinimidyl Ester) 500/506 Green 5 mg D2191 BODIPY FL NHS Ester (Succinimidyl Ester) 505/513 Green 5 mg D2184 Fluorescein-5-Isothiocyanate (FITC 'Isomer I') 494/518 Green 1 g F143 Alexa Fluor 488 TFP Ester 495/519 Green 3 x 100 µg A37570 25 mg A37563 Alexa Fluor 488 NHS Ester (Succinimidyl Ester) 495/519 Green 1 mg A20000 5 mg A20100 Alexa Fluor 488 Protein Labeling Kit 495/519 Green 1 kit A10235 Alexa Fluor 488 Microscale Protein Labeling Kit 495/519 Green 1 kit A30006 Alexa Fluor 488 Antibody Labeling Kit 495/519 Green 5-rxn kit A20181 APEX Alexa Fluor 488 Antibody Labeling Kit 495/519 Green 1 kit A10468 Zip Alexa Fluor 488 Rapid Antibody Labeling Kit 495/519 Green 1 kit Z11233 pHrodo iFL Green STP Ester, amine-reactive dye 505/520 Green 3 x 100 µg P36012 1 mg P36013 pHrodo iFL Green Microscale Protein Labeling Kit 505/520 Green 1 kit P36015 Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer 495/524 Green 5 mg O6149 Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 5-isomer 495/524 Green 5 mg O6147 Oregon Green 514 Carboxylic Acid, Succinimidyl Ester 511/530 Yellow 5 mg O6139 Alexa Fluor 514 NHS Ester (Succinimidyl Ester) 517/542 Yellow 1 mg A30002 Alexa Fluor 430 NHS Ester (Succinimidyl Ester) 434/539 Yellow 5 mg A10169 Pacific Orange Succinimidyl Ester, Triethylammonium Salt 400/551 Orange 1 mg P30253 Alexa Fluor 532 NHS Ester (Succinimidyl Ester) 531/554 Orange 1 mg A20001 5 mg A20001MP BODIPY 530/550 NHS Ester (Succinimidyl Ester) 534/554 Orange 5 mg D2187 Alexa Fluor 555 NHS Ester (Succinimidyl Ester) 555/565 Orange 3 x 100 µg A37571 1 mg A20009 5 mg A20109 25 mg A37564 Alexa Fluor 555 Protein Labeling Kit 555/565 Orange 1 kit A20174 Alexa Fluor 555 Microscale Protein Labeling Kit 555/565 Orange 1 kit A30007 Alexa Fluor 555 Antibody Labeling Kit 555/565 Orange 5-rxn kit A20187 APEX Alexa Fluor 555 Antibody Labeling Kit 555/565 Orange 1 kit A10470 Zip Alexa Fluor 555 Rapid Antibody Labeling Kit 555/565 Orange 1 kit Z11234 BODIPY 558/568 NHS Ester (Succinimidyl Ester) 558/569 Orange 5 mg D2219 BODIPY TMR-X NHS Ester (Succinimidyl Ester) 542/574 Orange/red 5 mg D6117 Alexa Fluor 546 NHS Ester (Succinimidyl Ester) 556/575 Orange/red 1 mg A20002 5 mg A20102 Alexa Fluor 546 Protein Labeling Kit 556/575 Orange/red 1 kit A10237 Alexa Fluor 546 Antibody Labeling Kit 556/575 Orange/red 1 kit A20183 BODIPY 576/589 NHS Ester (Succinimidyl Ester) 576/590 Red 5 mg D2225 pHrodo iFL Red STP Ester, amine-reactive dye 566/590 Red 3 x 100 μg P36011 1 mg P36010 Alexa Fluor 568 NHS Ester (Succinimidyl Ester) 578/603 Red 1 mg A20003 5 mg A20103 Alexa Fluor 568 Antibody Labeling Kit 578/603 Red 5-rxn kit A20184 APEX Alexa Fluor 568 Antibody Labeling Kit 578/603 Red 1 kit A10494 Alexa Fluor 568 Protein Labeling Kit 578/603 Red 1 kit A10238 Fluorescent dye labeling reagents and kits 61 Product Emission/excitation (nm) Emission color Quantity Cat. No. Amine-reactive BODIPY TR-X NHS Ester (Succinimidyl Ester) 589/617 Red 5 mg D6116 Alexa Fluor 594 NHS Ester (Succinimidyl Ester) 590/617 Red 3 x 100 μg A37572 1 mg A20004 Alexa Fluor 594 NHS Ester (Succinimidyl Ester) 590/617 Red 5 mg A20104 25 mg A37565 Alexa Fluor 594 Protein Labeling Kit 590/617 Red 1 kit A10239 Alexa Fluor 594 Microscale Protein Labeling Ki 590/617 Red 1 kit A30008 Alexa Fluor 594 Antibody Labeling Kit 590/617 Red 1 kit A20185 APEX Alexa Fluor 594 Antibody Labeling Kit 590/617 Red 1 kit A10474 BODIPY 630/650-X NHS Ester (Succinimidyl Ester) 625/640 Far red 5 mg D10000 Alexa Fluor 633 NHS Ester (Succinimidyl Ester) 632/647 Far red 1 mg A20005 5 mg A20105 Alexa Fluor 633 Protein Labeling Kit 632/647 Far red 5-rxn kit A20170 BODIPY 650/665-X NHS Ester (Succinimidyl Ester) 646/660 Far red 5 mg D10001 Alexa Fluor 647 NHS Ester (Succinimidyl Ester) 650/668 Far red 3 x 100 μg A37573 1 mg A20006 5 mg A20106 25 mg A37566 Alexa Fluor 647 Protein Labeling Kit 650/668 Far red 1 kit A20173 Alexa Fluor 647 Microscale Protein Labeling Kit 650/668 Far red 1 kit A30009 Alexa Fluor 647 Antibody Labeling Kit 650/668 Far red 5-rxn kit A20186 APEX Alexa Fluor 647 Antibody Labeling Kit 650/668 Far red 1 kit A10475 Zip Alexa Fluor 647 Rapid Antibody Labeling Kit 650/668 Far red 1 kit Z11235 Alexa Fluor 680 NHS Ester (Succinimidyl Ester) 679/702 Far red 3 x 100 μg A37574 1 mg A20008 5 mg A20108 25 mg A37567 Alexa Fluor 680 Protein Labeling Kit 679/702 Far red 1 kit A20172 Alexa Fluor 680 Antibody Labeling Kit 679/702 Far red 5-rxn kit A20188 Alexa Fluor 700 NHS Ester (Succinimidyl Ester) 702/723 NIR 1 mg A20010 5 mg A20110 Alexa Fluor 790 NHS Ester (Succinimidyl Ester) 702/723 NIR 100 μg A30051 Alexa Fluor 790 Antibody Labeling Kit 702/723 NIR 5-rxn kit A20189 Sulfhydryl-reactive Alexa Fluor 350 C5 Maleimide 346/442 Blue 1 mg A30505 BODIPY FL Maleimide (BODIPY FL N-(2-Aminoethyl))Maleimide) 410/455 Blue 1 mg B10250 Fluorescein-5-Maleimide 505/513 Green 25 mg F150 Alexa Fluor 488 C5 Maleimide 494/518 Green 1 mg A10254 Oregon Green 488 Maleimide 495/519 Green 1 mg O6034 Alexa Fluor 532 C5 Maleimide 495/524 Green 1 mg A10255 Alexa Fluor 555 C2 Maleimide 531/554 Green 1 mg A20346 BODIPY TMR C5 Maleimide 555/565 Orange 1 mg B30466 Alexa Fluor 546 C5 Maleimide 542/574 Orange/red 1 mg A10258 Tetramethylrhodamine-5-Maleimide, single isomer 556/575 Orange/red 1 mg T6027 Rhodamine Red C2 Maleimide 555/580 Orange/red 1 mg R6029 Alexa Fluor 568 C5 Maleimide 570/590 Red 1 mg A20341 Texas Red C2 Maleimide 580/605 Red 1 mg T6008 Alexa Fluor 594 C5 Maleimide 595/615 Red 1 mg A10256 Alexa Fluor 633 C5 Maleimide 590/617 Red 1 mg A20342 Alexa Fluor 647 C2 Maleimide 632/647 Far red 1 mg A20347 Alexa Fluor 680 C2 Maleimide 650/668 Far red 1 mg A20344 Alexa Fluor 750 C5 Maleimide 679/702 NIR 1 mg A30459 62 Product Emission/excitation (nm) Emission color Quantity Cat. No. Carboxyl-reactive Alexa Fluor 350 Hydrazide 346/442 Blue 5 mg A10439 Fluorescein-5-Thiosemicarbazide 494/518 Green 100 mg F121 Fluorescein Cadaverine 494/518 Green 25 mg A10466 Alexa Fluor 488 Hydrazide 495/519 Green 1 mg A10436 Alexa Fluor 488 Hydroxylamine 495/519 Green 1 mg A30629 Alexa Fluor 488 Cadaverine 495/519 Green 1 mg A30676 Qdot 525 ITK Carboxyl Quantum Dots 405/525 Green 250 µL Q21341MP Qdot 545 ITK Carboxyl Quantum Dots 405/545 Yellow 250 µL Q21391MP Qdot 565 ITK Carboxyl Quantum Dots 405/565 Orange 250 µL Q21331MP Alexa Fluor 555 Hydrazide 555/565 Orange 1 mg A20501MP Alexa Fluor 555 Cadaverine 555/565 Orange/red 1 mg A30677 5-TAMRA (5-Carboxytetramethylrhodamine), single isomer 555/580 Orange/red 10 mg C6121 Qdot 585 ITK Carboxyl Quantum Dots 405/585 Red 250 µL Q21311MP Qdot 605 ITK Carboxyl Quantum Dots 405/605 Red 250 µL Q21301MP 5-ROX (5-Carboxy-X-Rhodamine, Triethylammonium Salt), single isomer 580/605 Red 10 mg C6124 Texas Red Hydrazide, >90% single isomer 595/615 Red 5 mg T6256 Qdot 625 ITK Carboxyl Quantum Dots 405/625 Red 250 µL A10200 Qdot 655 ITK Carboxyl Quantum Dots 405/655 Far red 250 µL Q21321MP Qdot 705 ITK Carboxyl Quantum Dots 405/705 NIR 250 µL Q21361MP Qdot 800 ITK Carboxyl Quantum Dots 405/800 NIR 250 µL Q21371MP Chemoselective SiteClick Antibody Azido Modification Kit NA NA 1 kit S20026 Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling 494/518 Green 1 each C20027 Alexa Fluor 488 Azide 494/518 Green 0.5 mg A10266 Alexa Fluor 488 Alkyne 494/518 Green 0.5 mg A10267 Click-iT Alexa Fluor 488 sDIBO Alkyne 494/518 Green 0.5 mg C20020 Click-iT Plus Alexa Fluor 488 Picolyl Azide Toolkit 494/518 Green 1 kit C10641 SiteClick Qdot 525 Antibody Labeling Kit 405/525 Green 1 kit S10449 SiteClick Qdot 565 Antibody Labeling Kit 405/565 Orange 1 kit S10450 Alexa Fluor 555 Azide 555/565 Orange/red 0.5 mg A20012 Alexa Fluor 555 Alkyne 555/565 Orange/red 0.5 mg A20013 Click-iT Alexa Fluor 555 sDIBO Alkyne 555/565 Orange/red 0.5 mg C20021 Click-iT Alexa Fluor 555 sDIBO Alkyne for Antibody Labeling 555/565 Orange/red 1 each C20028 Click-iT Plus Alexa Fluor 555 Picolyl Azide Toolkit 555/565 Orange/red 1 kit C10642 SiteClick Qdot 585 Antibody Labeling Kit 405/585 Orange/red 1 kit S10450 SiteClick R-PE Antibody Labeling Kit 565/578 Orange/red 1 kit S10467 Tetramethylrhodamine (TAMRA) Azide 555/580 Red 0.5 mg T10182 Tetramethylrhodamine (TAMRA) Alkyne 555/580 Red 0.5 mg T10183 Click-iT pHrodo iFL Red sDIBO Alkyne for Antibody Labeling 566/590 Red 1 kit C20034 SiteClick Qdot 605 Antibody Labeling Kit 405/605 Red 1 kit S10469 Alexa Fluor 594 Azide 590/617 Red 0.5 mg A10270 Alexa Fluor 594 Alkyne 405/625 Red 0.5 mg A10275 SiteClick Qdot 655 Antibody Labeling Kit 405/655 Far red 1 kit S10453 Alexa Fluor 647 Azide 650/668 Far red 0.5 mg A10277 Alexa Fluor 647 Alkyne 650/668 Far red 0.5 mg A10278 Click-iT Alexa Fluor 647 sDIBO Alkyne 650/668 Far red 0.5 mg C20022 Click-iT Alexa Fluor 647 sDIBO Alkyne for Antibody Labeling 650/668 Far red 1 kit C20029 Click-iT Plus Alexa Fluor 647 Picolyl Azide Toolkit 650/668 Far red 1 kit C10643 SiteClick Qdot 705 Antibody Labeling Kit 405/705 NIR 1 kit S10454 SiteClick Qdot 800 Antibody Labeling Kit 405/800 NIR 1 kit S10455 63 Related handbooks and resources The Molecular Probes Handbook The most complete reference available on fluorescent labeling and detection, The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies contains information on over 3,000 reagents and kits representing the full range of our labeling and detection products. The 11th edition features extensive references, reorganized content, and new technical notes and product highlights. Download the free handbook at thermofisher.com/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 For Research Use Only. Not for use in diagnostic procedures. © 2018, 2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Hoechst is a trademark of Hoechst GmbH. EXT3222 0822 Learn more at thermofisher.com/proteincrosslinking