Immunoblotting

A three-step procedure used to separate proteins and subsequently identify a specific protein of interest.

Western blot is a powerful tool to detect and characterize a multitude of proteins. It involves 3 steps: (1) A protein mixture is separated through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS gel, and then transferred from the gel onto a solid support, usually nitrocellulose paper. (2) The membrane is flooded with a solution of antibody (Ab₁) specific for the desired protein. Only the band containing this protein binds the antibody, forming a layer of antibody molecules (although their position can't be seen at this point). The nitrocellulose paper is then washed to remove unbound Ab₁. (3) In the development step, the membrane first is incubated with a second antibody (Ab₂) that binds to the bound Ab₁. This second antibody is linked to alkaline phosphatase, which catalyzes a chromogenic reaction. The...