Western blots are SDS-PAGE gels taken one step further. SDS-PAGE is a process of separating proteins based on size. Proteins are denatured and loaded on to a polyacrylamide gel and an electric field is applied which causes smaller proteins to move through the gel towards the positive electrode faster than larger proteins. Once the proteins are sufficiently separated on the gel, they can be transferred out of the gel onto nitrocellulose or PVDF membrane which is the first step of performing a western blot. The membrane can be blocked with BSA or milk and then it is exposed to a primary antibody which binds one particular protein on the membrane. A secondary antibody binds the Fc region of primary antibody. The secondary antibody is conjugated to a enzyme that can catalyze a chemiluminescent reaction, such as horseradish peroxidase, When substrate is added to the membrane, the protein of interest can be visualized as a distinct band on the membrane. Typically, the membrane is exposed to film and the band is immortalized as a dark band on the film.
Because the primary antibody is specific to the protein of interest, Western blots can be used as an identity test. The molecular weight of the protein confirms the identity. PBL can perform western blot as a cGMP process, as part of a GLP study or as an R&D project.