Western Blot Procedures, Analysis and Purpose

There are many reasons you may need to determine the presence (or absence) and size of a given protein within a sample. Be it to detect or investigate disease, to determine the success or failure of genetic manipulation experiments or to identify the presence of potentially allergenic proteins in a food sample. To pinpoint your target protein, you will often need to distinguish it from a background of many others, some of which may share similar properties or size. The western blot is one such technique that offers these capabilities.

In this article, we discuss what western blots are, how they are performed, what they show us and how they differ to related techniques.

What is a western blot?

A western blot, sometimes called a protein immunoblot, is an antibody-based technique used to detect the presence, size and abundance of specific proteins within a sample. The technique was developed in 1979¹ by Harry Towbin and colleagues and later named the “western blot” due to the technique’s similarity to Southern blotting.²

Briefly, proteins in an aqueous sample are separated by electrophoresis. Following transfer to an appropriate membrane, the samples are probed using target-specific antibodies. These antibodies can be detected, and the size and abundance of the bound proteins evaluated in comparison to known standards or controls.

Western blot protocol

Figure 1 shows an overview of a western blot protocol.^{3, 4}

Figure 1: Overview of a western blot protocol. Credit: Technology Networks

Western blot gel

Before a western blot can be performed, the proteins in the sample must be separated. This is typically achieved by protein electrophoresis, such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE, which separates proteins based on their molecular weight or charge. The specific separation method chosen will depend on the aim of the analysis. For a clean image, samples are centrifuged to remove any solids, in order to load only the soluble fraction. If your protein of interest is in the insoluble fraction (e.g., cell membrane-bound proteins) investigate pretreatment methods to liberate and solubilize it first. Solids will impair the running of the gel and it is likely your protein of interest will remain in the stacking gel.

It is also important to load appropriate control samples and size marker ladders to enable interpretation of the final blot.

Loading control for western blot

It is essential, especially when trying to compare protein expression between different samples, to know how much sample has been loaded as this may not be apparent from the blot alone. For example, when assessing a blot, the band from one sample may appear twice as bright as another sample. This could mean that there is twice as much of the target protein in that sample, or it could mean that more sample or a more concentrated sample has been loaded in one lane than the other. Running a duplicate protein gel and developing with Coomassie stain⁵ can help to remove this uncertainty as it will show the amount of total protein⁶ in each sample lane and can reveal any loading inconsistencies. Detecting expression of a ubiquitous protein that should be even between all of your samples, such as actin in whole cell and cytoplasmic samples, can also be used as a loading control and helps to ensure consistent transfer of protein samples to the membrane. However, this type of control can be problematic when comparing models in which “control” proteins are differently expressed, such as degeneration models.^{6, 7}

Protein transfer (blotting)

Proteins must be transferred from the protein gel to an appropriate membrane (typically nitrocellulose or polyvinylidene difluoride (PVDF)) to facilitate antibody probing. A number of techniques can be used for transfer, including capillary transfer, diffusion transfer and vacuum blotting, but by far the most common due to its speed and efficiency is electroblotting⁸ (also called electroelution or electrophoretic transfer). Here, the protein gel is sandwiched against the transfer membrane and an electrical current is applied. Proteins from the gel are carried across and attach to the membrane tightly.

Within electroblotting, there are also multiple strategies for transfer, known as wet, semi-dry and dry transfer. Wet transfer is efficient and offers flexible buffering but is time-consuming. Semi-dry transfer is quicker and still offers flexibility but is less efficient than wet transfer for large proteins. Dry transfer is efficient and quick but offers less flexibility than the other methods.

Transfer efficiency can be assessed prior to probing using a removable stain such as Ponceau S.

Western blot blocking

Due to the high affinity of blotting membranes for proteins, after transfer it is important to block any remaining binding sites to prevent subsequent non-specific binding of the assay detection antibodies. This is achieved by incubating the membrane with a proteinaceous liquid such as milk or serum.

Western blot washing

Following blocking, it is important to wash the membrane between each step to remove excess or unbound reagents. Insufficient or uneven washing can lead to poor quality/patchy blots and high background. However, over washing can diminish the target signal so it is important to optimize the number and duration of wash steps. Ensure the membrane is well covered with an appropriate buffer and apply gentle agitation to wash the membrane evenly without damaging it. Commonly used buffers include tris-buffered saline (TBS) and phosphate-buffered saline (PBS), often with the inclusion of Tween 20 (TBST and PBST).

Western blot antibody

While it is possible to use direct detection (a single antibody that recognizes the target and is detectable) for a western blot, more often an indirect method is applied (Figure 2). Here, a primary antibody is used to probe the membrane and bind any target protein present. Then, a secondary antibody is used that binds the primary antibody and is detectable. As with all steps, optimization,⁹ in this case choosing the “right” antibody and determining the optimal concentration, is key to a good blot.

Figure 2: Direct versus indirect western blot, the example used shows chemiluminescent detection. Credit: Technology Networks

Western blot secondary antibody

When an indirect detection assay is used, a secondary antibody will need to be applied after washing excess unbound primary antibody off the membrane. The secondary antibody should be specific to the species of the primary antibody (e.g., mouse anti-rabbit if the primary antibody was derived from a rabbit) and possess the necessary conjugate for the chosen detection method.

Western blot analysis

There are multiple methods for detection and subsequent visualization of western blots including colorimetric, chemiluminescent, fluorescent and radioactive detection, summarized in Figure 3.

Figure 3: Examples of colorimetric, chemiluminescent, radioactive and fluorescent detection using an indirect method. 

Both colorimetric and chemiluminescent detection require conjugation of an enzyme to the detection antibody and are considered very sensitive techniques. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used enzymes, with HRP generally favored due to its stability, amenability to most conjugations and low cost. During detection, a substrate is added to the membrane, which is acted on by the conjugated enzyme, bringing about a chemical change. If performing colorimetric detection, a chromogenic substrate is chosen that will produce a change that can be visualized and imaged directly. However, prompt imaging of the blot is important as colors will fade as the blot dries. In chemiluminescent detection,¹⁰ the signal produced only lasts as long as the reaction between the enzyme and substrate is occurring (typically 1–24 hours). During this time, the signal can be recorded by exposing X-ray film or using digital imaging to make a permanent record.

In fluorescent detection, the detection antibody is conjugated with a fluorophore rather than an enzyme. When light of a specific wavelength is shone on them, they become excited and emit light of a different specific wavelength. This can then be captured visually using digital imagers, such as an avalanche photodiode (APD), photomultiplier tube (PMT) or charge-coupled device (CCD) camera. While specialist equipment is required to undertake the excitation and detection steps, there is no substrate step in fluorescent detection, shortening the protocol. It is also possible to multiplex fluorophores¹¹ within a western blot assay.

Radioactive detection, where a radioisotope is conjugated to the detection antibody and the emitted radiation is detected on X-ray film, was used extensively in the past. However, the technique requires special handling to protect personnel from the radiation, is expensive and has a limited shelf life due to radioactive decay. Therefore, the technique has mostly been replaced in favor of other available detection methods.

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Western blot stripping buffer

Stripping is the process of removing primary and secondary antibodies from a western blot membrane. This can be helpful if you wish to investigate more than one target protein on a particular blot rather than running multiple blots, for example where samples are extremely valuable or scarce.

Be aware that stripping may also remove some protein from the membrane so quantitative comparisons cannot be made before and after stripping and checks for the absence of a protein should not be made on a stripped membrane.

PVDF membranes¹² are recommended if you intend to strip your blot as they are better at retaining proteins after stripping than nitrocellulose. Colorimetric detection¹³ also leaves a permanent stain so chemiluminescent detection is favored for this application.

If possible, start with mild stripping buffers that strip with low pH to minimize sample loss and only try more stringent stripping via detergent or heat if this is unsuccessful.

Western blot results and quantification

When using X-ray film to obtain results from your western blot experiment, it is often necessary to expose several films to optimize exposure and developing time. Not long enough and sample bands may be faint or not visible at all. Too long and the background signal becomes too strong, bands merge together and the resultant blot is very dark and hard to interpret. Controls or size markers that are not balanced in concentration with the target can also be problematic, with one becoming overexposed before the other is sufficiently visible. Films may then be digitized for further interrogation using analysis software.^{14, 15}

Detection methods utilizing X-ray film are considered qualitative or semi-quantitative. Comparison of bands in sample lanes to controls and size markers can help to establish the presence or absence of a target within a sample. However, if quantitation¹⁶ is required, digital imaging with CCD camera-based devices for example, offers a better option due to their larger dynamic range, greater sensitivity and higher resolution. Exposure times can be fine-tuned without the need for repeated time-consuming X-ray film exposures and developments, optimizing the signal-to-noise ratio. This can then easily be fed into software that can analyze¹⁷ and compare the data.

Western blot troubleshooting

There are many opportunities within a western blot protocol for things to go wrong, here we highlight a few common problems and provide potential solutions.^{18, 19}

• Insufficient separation/poor positioning of protein bands on gel (and subsequently blot) – Optimize the protein gel percentage and run time to suit your target.
• Nothing on your blot, not even the ladder – Ensure the blotting kit is put together in the right order and the current is running in the right direction otherwise your sample will blot into the buffer.
• Blot appears messy/patchy – Ensure no bubbles remain between the gel and membrane when setting up the transfer. Make sure the membrane is fully submerged and gently mixed during incubation and wash steps to create an even result. Use fresh blocking reagent.
• High background – Blocking or washing may be insufficient.
• Results of control samples are inconsistent with expected outcome – Optimize antibody choice/concentration to minimize non-specific/off-target binding. Check your experimental assumptions and concepts. Are your expectations incorrect?
• Bands are hard to see on the final blot (too dark or light) – Optimize developing times in the detection phase to increase or decrease signal strength. If bands appear too light, it may also indicate over washing. Optimizing antibody concentrations and considering your choice of antibody may also help.
  

What's the difference between a Southern blot vs western blot?

Whereas a western blot is designed to detect specific proteins, a Southern blot,² developed in 1975 and named after its inventor Edwin Southern,²⁰ is used to detect specific DNA sequences. As with a western blot, samples are separated by electrophoresis and transferred onto a membrane. However, DNA fragments with sequences complimentary to the region you wish to detect are then used to probe the membrane prior to detection.

What's the difference between a northern blot vs western blot?

Northern blotting,² developed in 1977 by James Alwine, David Kemp and George Stark,²¹ is used to detect specific mRNA sequences. As with western and Southern blotting, samples undergoing northern blotting are separated by electrophoresis and transferred to a membrane. cDNA fragments with sequences that are complimentary to the region of interest are then used as probes prior to detection.

What's the difference between a western blot vs SDS-PAGE?

SDS-PAGE is an electrophoresis technique used to separate proteins according to their molecular mass, whereas a western blot is the whole process required to detect the presence of a specific protein. SDS-PAGE is often the electrophoresis technique chosen within a western blot protocol as a means to separate out all the proteins within a sample prior to blotting.

Western blot purpose

There are many reasons why detecting the presence, size and abundance of target proteins within a sample may be desirable and these reasons can span a multitude of scientific disciplines. As well as the presence or absence of a target, the technique enables evaluation of:²²

• Protein–DNA interactions – Attachment of DNA binding proteins to specific DNA sequences is vital to the process of transcriptional regulation. Southwestern blotting²³ (similar to western blotting except the membrane is probed with DNA) can be used to identify transcription factors²⁴ in gene regulation studies.
  
  
• Protein–protein interactions – These are vital for many essential cellular processes. Detection of protein–protein interactions, using a variation of western blotting known as far-western blotting,^{25, 26} can help to elucidate cellular function and dysfunction.
  
  
• Post-translational modifications (PTMs) – PTM’s impact protein folding and consequently function, expanding proteome diversity. However, aberrant PTM’s have been associated with disease. Consequently, their study is of great interest to researchers and western blot provides one such tool with which to do this.
  
  
• Protein isoform detection – Proteins may be expressed in differing isoforms in different cellular states with varying activities or targets. Alternatively, they may require cleavage to become activated.
  
  
• Antibody characterization – When antibodies are produced as tools or therapeutics, their validation and characterization²⁷ is vital to ensure correct performance and safety. As an antibody-based method, western blotting is a useful technique in this process.
  
  
• Epitope mapping – Understanding how and where antibodies bind their target protein is valuable for research, diagnostic and therapeutic purposes. There are many tools used towards this goal and thanks to its specificity, western blotting²⁸ is one.
  
  
• Subcellular protein localization – Performing western blot analysis of different cellular fractions allows the location of target proteins in the cell to be determined. Single-cell western blotting²⁹ has offered great insights in this field and overcomes some of the antibody cross-reactivity issues experienced by other single-cell assays.

Some common applications are discussed below.

Determine if a protein is expressed

Assessing genome data can tell you if the organism in question has the potential to produce certain proteins. Looking at the transcriptome will tell you if the gene in question is turned on, but it’s not until you can detect the actual protein that you can tell if it is actively being expressed.³⁰ When performing experimental genomic manipulations this can be very helpful to determine if the changes made at the genomic level are reflected by the expected changes at the protein level³¹ (e.g., expression of a novel protein, truncation or loss of expression compared to a wild-type strain, is it expressed in the expected tissue or location?).

Detect tagged proteins

When performing genetic manipulations, it is possible to engineer and add tags, such as His-tag and GST-tag.³² These make the protein to which they are attached easy to find³³ in samples from the species or system being studied, providing information on the location and fate of the protein in question. For example, is a membrane protein cleaved from the cell membrane under certain conditions. This can also be useful when checking experimentally produced recombinant proteins.³⁴

Mapping changes over time or between groups

Western blotting on time course experiment samples can provide information about protein level changes over time, for example during different cell cycle phases. Equally, when comparing samples from different disease or treatment groups, it can highlight variations at the protein level that may indicate an underlying cause of disease or influence therapeutic effect.

Overall, western blotting is not considered a particularly quantitative technique compared to the likes of PCR. However, the direct detection of proteins it offers means it has found great utility in confirmatory disease diagnoses.

Biomarkers of non-infectious disease

Differing protein isoforms may act as biological markers of pathological processes, for example in cancers.³⁵ Autoantibodies, indicative of autoimmune disease, may also be detected. While mass spectrometry is often used for their detection, antibody-based techniques like western blot are still used to confirm³⁶ the findings of high-throughput methods.

Infectious disease diagnosis

Western blot has been used to confirm cases of diseases such as human immunodeficiency virus (HIV),³⁷ Lyme disease,^{38, 39} bovine spongiform encephalopathy (BSE)⁴⁰ and aspergillosis.⁴¹ However, it is time consuming compared to techniques such as PCR and has been replaced by alternative assays in a number of cases including HIV testing.