Western Transfer, also known as Western Blotting, is a rapid immunoblotting technique for identifying the presence of a particular protein in a complex mixture of proteins such as cell lysates or sera. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, and the ability of immunochemical reagents to interact specifically with a given protein antigen.
A typical Western-transfer protocol (e.g. for the detection of protein X) includes the following steps: [1] The proteins in the tested solution are separated into distinct bands by SDS-PAGE, [2] The size-separated proteins are transferred from the polyacrylamide gel to a nitrocellulose membrane (or another suitable matrix). [3] The membrane containing 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-X antibody) that forms a specific immune complex with Protein-X, [d] additional wash solution to remove any unbound antibody, [e] an enzyme-linked antibody that binds specifically to the Fc region of the anti-Protein-X antibody, [f] additional wash solution to remove any unbound enzyme-linked antibody, and finally, [g] a substrate solution, which in the presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rendering the Protein X band visible (Figure III-1).
Transfer Protocol:
Development (Immunostaining) Protocol: