Overview & Resources
Western blot, or western blotting, is an analytical technique used to perform protein analysis. Specifically, western blotting, also referred to as immunoblotting, allows for the visualization and study of target proteins within a mixture of proteins.
The technique involves using gel electrophoresis to separate the sample’s proteins, which are transferred out of the gel to the surface of a membrane. After, the membrane is exposed to an antibody that is specific to the target protein. The antibody binds to the target protein, and the binding is detected using labeled antibodies, or conjugated molecules.
This method of detection is considered an indirect method, in comparison to direct methods where the protein of interest in the complex protein mixture is detected using an enzyme- or fluorophore-conjugated primary antibody.
For the indirect detection method, labels can include a variety of fluorescent probes and enzyme conjugates, including Invitrogen Alexa Flour, DyLight flourophores, horseradish peroxidase (HRP), and alkaline phosphatase (AP). Biotin may also be used for detection.
From separating proteins, transferring to a membrane, and blocking nonspecific sites to picking a washing buffer formulation and primary and secondary antibodies, there are several key parts to the western blot method.