Western Blotting (also known as immunoblotting) is a technique used in biomedicine to isolate and identify a protein of interest. Western Blotting involves the use of antibodies to bind to the protein (primary antibodies) and then to identify through bioluminescence (secondary antibodies). It is possible to not only identify the protein with the fluroescent label but we can also determine the quantity of the protein.
Protocol for Western Blotting:
The [protein] is run on an SDS-polyacrylamide gel. To enable the proteins to be detected the gel is transfered onto a polmer sheet, such as nitrocellulose, it is simply pressed against the gel transfering the protein to the sheet. This nitrocellulose sheet is then exposed to the primary antibody that is specific to the protein that we want to identify. A different method to adding secondary antibody is the ELISA method where an enzyme on the second antibody will produce a coloured product.
Non-fat dried milk is often used in Western blotting as it binds to the membrane, preventing non-specific antibody binding and it reduces background signal.