The western blot test is an immunological technique that separates proteins based on their size and then detects binding by specific antibodies. This method is used to confirm a suspected antibody-antigen interaction and is often used in conjunction with other immunologic tests such as immunohistochemistry (IHC).
During SDS-PAGE, samples are loaded onto an acrylamide gel and protein sizes are separated using electrophoretic mobility as smaller proteins move through the gel faster than larger ones. The proteins are then transferred to a nitrocellulose or PVDF membrane that binds proteins. The membrane can then be “probed” with a secondary antibody that has been covalently linked to an enzyme. The antibody-enzyme complex binds to the target protein on the blot and results in a colorimetric signal. The soluble dye that is produced by the reporter enzyme in this two-step detection system may be visualized using a variety of techniques, including electrochemiluminescence, fluorescent and radioactive labeling.
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If a blot produces no bands, there are a number of reasons why this can occur. The primary antibody could be inappropriate for the antigen being tested, or the concentration of the antibody may be too low to produce a signal. Additionally, if the buffers used for the assay are not optimized, the protein sample will not be correctly separated and will not bind to the secondary antibody.
Another common reason for a negative result is that the antibodies are not binding to their target protein. This is most likely to be the case if multiple proteins are bound by an antibody. For example, the heparan sulfate proteoglycan antibody to syndecan-4 binds to numerous proteins of varying molecular weights and is not specific for a prion protein, such as those found in bovine spongiform encephalopathy (BSE; also known as mad cow disease). In these instances, biochemical and/or histochemical analysis of the protein can help confirm the antibody’s binding activity to its intended epitope.
