40.1 Western Blotting Assay

It is the most popular and widely used method to identify and determine the size of an unknown protein. The mixtures of protein are first subjected to an SDS-PAGE, so that the proteins can be separated based on their size. The gel is then transferred in to a nitrocellulose membrane in a way to represent its exact replica. Protein band on the membrane is then detected by binding an antibody specific to that protein followed by a labeled secondary antibody which binds to the first antibody. The second antibody is labeled with an enzyme that generates chemiluminescent signals which can be detected over the x-ray film. The sensitivity and specificity of western blot can be increased by purifying the proteins first with Immunoprecipitation assay.

 

* Interesting Facts

The scientist who first blotted the DNA over the membrane by capillary transfer was Edwin Southern, and hence named Southern blotting . By analogy, transfer of RNA over the membrane is called Northern blotting and transfer of protein is called Western blotting .

There is no Eastern blot???

 

Figure 39.1 Schematic representation of western blotting assay: