BioTechniques Examines Western Blotting: Technique, Troubleshooting and History

Western blotting may come with its challenges, but new techniques and technologies can help researchers overcome these.

The well-established biochemical technique western blotting (or immunoblotting) detects proteins in complex samples and is key to several applications in research and disease diagnoses. The technique has been around for over four decades, during which researchers have utilized it in thousands of studies. Researchers use the western blot to determine a protein’s molecular weight, quantity and post-translational modifications, making the technique a powerful tool for tracking protein expression. Its benefits are particularly relevant to pain management and cancer therapies.

As the western blot and its associated technologies evolve, we can expect to see developments in the technique’s miniaturization, automation, and speed/ease of use, not to mention the standards designed to improve reproducibility. The life sciences journal BioTechniques publishes a wealth of material about the development of western blotting, particularly advancements in technique and troubleshooting technologies.

Western Blotting Technique

Three key phases make up the western blotting technique.

1) Separate the Proteins in the Sample. Researchers can achieve this through gel electrophoresis. They might separate the proteins by many factors, like molecular weight, electric charge, or isoelectric point. The treatment of the sample and the nature of the gel dictate this decision. The most common type of gel electrophoresis, SDS-PAGE, utilizes polyacrylamide gels and buffers loaded with sodium dodecyl sulfate. This process creates denatured proteins that are separated according to molecular mass.

2) Transfer the Proteins to a Support. Researchers make the separated proteins accessible for antibody detection by transferring them from the gel to a membrane. The most common process for this transfer is electroblotting: an electric current pulls the proteins from gel to membrane without disrupting their organization.

3) Visualize the Target Protein Using Primary and Secondary Antibodies. Researchers ‘block’ the membrane to prevent non-specific antibody binding. Then, they stain the membrane with primary antibodies that are specific to the target protein and add a tagged secondary antibody that binds to the primary antibody. Researchers can visualize the tagged antibodies through one of many methods. For example, immunofluorescence enables indirect detection of the target protein.

Troubleshooting Western Blotting Challenges

Challenges can arise at any point during the western blotting process, which has a seemingly random tendency to fail. Usually, these challenges fall into one of five groups.

1) Unusual or Unexpected Bands  These are usually caused by protease degradation, which produces bands in unexpected positions. Researchers can avoid unusual bands by altering the antibody and by using a fresh sample that has been kept on ice.

2) No Bands  These are usually caused by a problem with the antibody, antigen, or buffer. The band won’t show if the researcher uses an improper primary or secondary antibody or an inappropriate concentration of an antibody. A low concentration or absence of the antigen can also result in no bands. A researcher can use an antigen from another source to confirm whether the problem lies with the sample or with other elements (like the antibody). Alternatively, prolonged washing and/or contaminated buffers can result in no bands.

3) Faint Bands or Weak Signal  These are usually caused by a low concentration of the antibody or the antigen. Researchers can try increasing the exposure time to make the band clearer. Alternatively, non-fat dry milk can mask the antigen, in which case researchers should use bovine serum albumin (BSA) or reduce the amount of milk used.

4) A High Background on the Blot  This is often caused by a high concentration of the antibody, which may bind to PVDF membranes. Alternatively, the buffers may be too old. Researchers can increase the washing time to decrease the background. Another possible reason for high background is that the exposure may be too high. In this case, researchers should check different exposure times to find the optimum time.

5) Patchy or Uneven Spots on the Blot  This is usually caused by improper transfer. Air bubbles trapped between the gel and membrane will appear darker on the film. Researchers should wash the background and use a shaker for incubation to avoid uneven agitation. Alternatively, antibodies binding to the blocking agents can also cause spots on the blot. In this case, researchers should try another blocking agent or filter the blocking agent to remove contaminants. Thirdly, aggregation of the secondary antibody can cause spots. Researchers can centrifuge and filter the antibody to eliminate the secondary antibody.

Emerging Technologies

The good news is that new technologies are emerging that eliminate or reduce the impact of the western blot’s challenges. For example, while researchers previously needed to stain and de-stain gels and blots, new Stain-Free technology has made this redundant. Bio-Rad’s Stain-Free technology removes the need to stain gels and membranes. Instead, the automated system confirms the successful completion of each stage, like electrophoresis and blotting. This means that researchers don’t need to apply costly detection agents and antibodies to blots that have already failed. Researchers can also use the Stain-Free blot to measure the protein present in each lane. This eliminates issues that can otherwise arise from errors in inconsistent gel-loading.

Other emerging technologies include the Trans-Blot Turbo system, which can transfer proteins to a membrane in as few as three minutes, and the ChemiDoc Imaging system, which can detect up to three proteins at the same time, eliminating the need to strip and reprobe.

The History of Western Blotting

The term ‘western blotting’ is a play on the term ‘Southern blotting’, a technique invented by Edwin Southern in 1975. Southern’s technique uses electrophoresis to separate pieces of DNA based on size. Researchers can transfer this DNA to a membrane and apply a hybridization process using a radiolabeled DNA probe to detect a specific DNA sequence and analyze its identity, size, and quantity. A couple of years later, in 1977, scientists James Alwin, David Kemp, and George Stark coined a similar technique, northern blotting, which detects specific RNA molecules in RNA samples.

Then, in 1979, postdoctoral researcher W Neal Burnette tried to combine SDS-PAGE electrophoresis with a radioimmunoassay but couldn’t visualize the interactions between the separated proteins and the antibodies in polyacrylamide gels. So, he followed the principles of the northern blot and created a solid-phase replica of the gel. He discovered that electrophoresis made it possible to blot proteins from SDS-PAGE gels onto nitrocellulose paper, which was more effective than chemically modified paper. He then used [125I] -labelled protein A to bind antibody-antigen complexes, eliminating the need for a second radiolabeled antibody.

Blocking non-specific binding sites of the protein A and the antibody to nitrocellulose paper enabled Burnette to generate clear radiographic images of antibody-specific antigens. The name ‘western blotting’ refers to the U.S. west coast location where Burnette developed the technique.

About BioTechniques

The peer-reviewed journal BioTechniques publishes open-access updates in life sciences methodologies, techniques, and instrumentation that prove invaluable to industry experts, lab researchers, other scientific professionals, and students. Many of these individuals make the most of both the print journal and BioTechniques’ expansive online community, where users benefit from free webinars, podcasts, eBooks, and articles and engage in meaningful discussions that pave the way for a rich future in science and medicine.