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Western Blotting

Biointron 2024-01-20 Read time: 3 mins
Image credit: DOI: 10.1016/B978-0-12-375156-0.00010-2

Western blotting, also known as immunoblotting, is a powerful analytical technique used to identify specific proteins within a complex mixture extracted from cells. The process involves several steps: protein extraction, gel electrophoresis, protein transfer, blocking, antibody incubation, and signal detection. This method uses antibodies to selectively target and bind to the protein of interest.1

Antibodies are the key component in western blotting, as they can affect accuracy, reproducibility and specificity of the results. Because of their importance, the International Working Group for Antibody Validation (IWGAV) was created to develop guidelines to ensure antibody reproducibility.2 These guidelines were used to score human protein targets on Antibodypedia, an antibody reagent portal, as well as the Human Protein Atlas.

There are various applications for western blotting, from scientific research to clinical-based uses. In research, western blots are often used to detect: 

  • Protein isoforms

  • Protein-protein interactions

  • Protein-DNA interactions

  • Post-translational modifications

  • Subcellular localizations

  • Epitope mapping

  • Antibody development and characterization3

For clinical applications, western blots are particularly useful as diagnostic tools to confirm infectious and non-infectious disease diagnosis. For example, with Lyme disease, western blotting can confirm ELISA test results, or be paired with penalized linear discriminant analysis in order to differentiate early and late Lyme disease patients.4,5 

However, since western blotting can be error-prone, training is necessary to ensure the proper statistical design, normalization method, valid reference proteins, and selection of valid antibodies.3

At Biointron, we are dedicated to accelerating your antibody discovery, optimization, and production needs. Our team of experts can provide customized solutions that meet your specific research needs. Contact us to learn more about our services and how we can help accelerate your research and drug development projects. 


  1. Mahmood, T., & Yang, P. C. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences, 4(9), 429.

  2. Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., Rimm, D. L., Rodriguez, H., Hiltke, T., Snyder, M., & Yamamoto, T. (2016). A proposal for validation of antibodies. Nature Methods 2016 13:10, 13(10), 823–827.

  3. Meftahi, G. H., Bahari, Z., Zarei Mahmoudabadi, A., Iman, M., & Jangravi, Z. (2021). Applications of western blot technique: From bench to bedside. Biochemistry and Molecular Biology Education, 49(4), 509–517.

  4. Mavin, S., Evans, R., Cornulier, T., & Bowman, A. S. (2018). The development of an IgG avidity Western blot with potential to differentiate patients with active Lyme borreliosis from those with past infection. Journal of Microbiological Methods, 146, 71–76.

  5. Aguero-Rosenfeld, M. E., Nowakowski, J., McKenna, D. F., Carbonaro, C. A., & Wormser, G. P. (1993). Serodiagnosis in early Lyme disease. Journal of Clinical Microbiology, 31(12), 3090–3095.

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