The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract.[1] Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.[2]
Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize.[1] A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off.[3] A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.[3]
Other related techniques include dot blot analysis, quantitative dot blot, immunohistochemistry and immunocytochemistry, where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA).
The name western blot is a play on the Southern blot, a technique for DNA detection named after its inventor, English biologist Edwin Southern. Similarly, detection of RNA is termed as northern blot.[4] The term "western blot" was given by W. Neal Burnette in 1981,[5] although the method itself was independently invented in 1979 by Jaime Renart, Jakob Reiser, and George Stark at Stanford University,[6] and by Harry Towbin, Theophil Staehelin, and Julian Gordon at the Friedrich Miescher Institute in Basel, Switzerland.[7] The Towbin group also used secondary antibodies for detection, thus resembling the actual method that is almost universally used today. Between 1979 and 2019 "it has been mentioned in the titles, abstracts, and keywords of more than 400,000 PubMed-listed publications" and may still be the most used protein-analytical technique.[8]
^ abMahmood T, Yang PC (September 2012). "Western blot: technique, theory, and trouble shooting". North American Journal of Medical Sciences. 4 (9): 429–434. doi:10.4103/1947-2714.100998. PMC 3456489. PMID 23050259.
^Begum H, Murugesan P, Tangutur AD (June 2022). "Western blotting: a powerful staple in scientific and biomedical research". BioTechniques. 73 (1): 58–69. doi:10.2144/btn-2022-0003. PMID 35775367. S2CID 250175915.
^ abMishra M, Tiwari S, Gomes AV (November 2017). "Protein purification and analysis: next generation Western blotting techniques". Expert Review of Proteomics. 14 (11): 1037–1053. doi:10.1080/14789450.2017.1388167. PMC 6810642. PMID 28974114.
^Cite error: The named reference Stark1977 was invoked but never defined (see the help page).
^Cite error: The named reference Burnette1981 was invoked but never defined (see the help page).
^Cite error: The named reference Renart1979 was invoked but never defined (see the help page).
^Cite error: The named reference Towbin1979 was invoked but never defined (see the help page).
^Cite error: The named reference Moritz2020 was invoked but never defined (see the help page).
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