Definition of Western Blotting

Western blotting is a technique that is used in analyzing protein molecules. It is also known as immunoblotting as an antibody is employed that specifically detects the antigen. The target protein can be detected and identified in a complex mixture of proteins because of the specificity of the interaction between the antibody and the antigen.


The qualitative as well as semi-quantitative data regarding the protein of interest can be generated by western blot. In the first step of western blot, the macromolecules present in the sample are separated by the technique of gel electrophoresis. After the separation, they are transferred to the nitrocellulose or PVDF membrane.


The membrane is then blocked so that no non-specific binding of the antibodies could occur. After this, the membrane is incubated with primary and secondary antibodies. It is then treated with a chemiluminescent substrate and the light output is captured with the help of an X-ray film.


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