Western Blot Procedure

1.	A protein sample is subjected to electrophoresis on an SDS-polyacrylamide gel. The sample can be a whole cell lysate or a fractionated sample following differential centrifugation, immunoprecipitation etc.
2.	Electroblotting transfers the separated proteins from the gel to the surface of a nitrocellulose membrane. Thus, the  nitrocellulose membrane is imprinted with the same protein bands as the gel, and the transferred proteins are more accessible for further treatment.
3.	The blot is incubated with a generic protein (such as milk proteins or BSA) which binds to any remaining sticky places on the nitrocellulose. This helps to minimize background signals.
4.	An antibody that is specific for the protein of interest (the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen.  Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules (but their position can't be seen at this point).
5.	Following several rinses for removal of non-specifically bound Ab1, the Ab1-antigen complex on the nitrocellulose sheet is incubated with a second antibody (Ab2), which specifically recognizes  the Fc domain of the primary antibody and binds it. Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex (as illustrated here).

• Movie accompanying the book "Molecular Cell Biology".