How likely is it to detect different isoforms of a protein using a monoclonal antibody in a Western Blot experiment?
Hello Rosario Rox
Most human genes can produce multiple protein isoforms. Western Blot detects not only the expected band but also additional band(s) on the membrane. In reality, both are common as Western Blot detects additional band(s) at wrong position(s) on the membrane as well as it can detect only the expected band. In the former situation, it is a common but hardly mentioned practice for the researchers to assume, although without supporting evidence, that those bands at wrong positions are non-specific proteins and should be cut off, presenting in the publications only the band of interest. It is very often that antibody supplier companies are blamed for selling not specific enough antibodies.
Although primary antibodies that recognize only a single isoform are useful, those that recognize multiple isoforms and thus seemingly are not specific enough may provide us with a more global picture of the protein products of the gene studied. For this reason, one should retain and present all bands appearing on the Western Blot membrane unless there is tangible evidence proving that the additional band(s) are non-specific.
To detect different isoforms of a protein, you may use isoform-specific antibodies that allow relative quantification of expression on Western Blot. For well-studied protein families, isoform-specific antibodies can usually be purchased. However, batch-to-batch variation in antibody performance as well as recognition of unspecific binding partners sometimes hamper precise protein identification through this method. Furthermore, when studying newly identified isoforms or lesser studied protein families, there are often no monoclonal, isoform-specific antibodies available.
If monoclonal antibodies are unavailable, the use of polyclonal antibodies should be assessed carefully as larger immunogens increase the likelihood of producing signals from various family members. If the proteins do not vary in size, their distinction is impossible.
Best.
Dear Rosario Rox,
You may want to look at the link below:
https://bitesizebio.com/34400/primary-and-secondary-antibodies-immunoblot-western-blotting/
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If the antibody was raised against the conserved domain (present intact in all isoforms) and if they are substantially different in molecular sizes (at least 10-20 kDa difference) isoforms could be detected by Western blotting. Otherwise, you need specific antibody raised against the unique domain (distinct in all isoforms) for a specific isoform of the protein for detection by Western blotting.
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