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:
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
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References:
Mahmood, T., & Yang, P. C. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences, 4(9), 429. https://doi.org/10.4103/1947-2714.100998
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. https://doi.org/10.1038/nmeth.3995
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. https://doi.org/10.1002/BMB.21516
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. https://doi.org/10.1016/J.MIMET.2018.02.002
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. https://doi.org/10.1128/JCM.31.12.3090-3095.1993
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