Western blotting: is used to identify a specific protein in a complex mixture of proteins. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, adn the ability of immunochemical reagents to interact specifically with a given protein antigen. A typical Western-blotting protocol incluces the following steps:
(1) The mixture is first electrophoretically separated on a polyacrylamide slab gel in the presence of SDS.
(2) The protein bands are then transferred to a nitrocellulose membrane by electrophoresis.
(3) The membrane contianing the protein bands is serially incubated with (a) a suitable blocking reagent to prevent non-specific protein binding, (b) a wash solution to rinse any unbound blocking reagent (c) a probing antibody (anti-protein-of interest antibody) that forms a specific immune complex with Protein-of interst, (d) additional wash solution to remove any unbound antibody, (e) an enzyme-linked that binds specifically to the Fc region of the anti-protein-of interest antibody, (f) additional wash solution to remoe any unbound enzyme-lined anntibody and finally, (g) a substrate solution, which int he presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rending the Protein of interest band visible.