Western blot is used in research to separate and identify proteins. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. Once the proteins are separated by size through gel electrophoresis, they are then transferred to a membrane producing a band for each protein. It is important to include a blocking buffer after transferring as it prevents antibodies from binding to the membrane nonspecifically.
A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the membrane. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background interference and improving the signal-to-noise ratio.
Based on the characteristics of antibody-antigen reactivity, we know that the blocking solution should block all unbound sites without replacing the target protein on the surface, without binding to target protein epitopes, and without cross-reacting with antibodies or detection reagents.
Common Base Buffer:
Blocking buffers are composed of a salt solution, with or without detergent, and a blocking agent.
Common blocking agents:
The most commonly used blocking solution is Bovine Serum Albumin (BSA), and the single component is suitable for most situations. The primary role of BSA is to prevent non-specific binding by blocking the leftover spaces on the membrane. If the immunogen of the antibody being used is conjugated to BSA, however, there may be non-specific binding. Since BSA has strong immunogenicity, in production many antibodies against BSA can be produced. If the antibody was conjugated to BSA block, use casein or non-fat milk to avoid cross-reaction.
Non-Fat Dried Milk
The biggest advantage of non-fat dried milk is that it is cheap, but due to its relatively complex composition, the scope of application is narrower. Targeting phosphorylated protein may result in the increased background and, because of its biotin content, cannot be used with biotin-labeled antibody systems. Non-fat dried milk contains small amounts of biotin and alkaline phosphatase residues, which can also lead to high background or increased background levels when using the biotin-avidin system and alkaline phosphatase-conjugated secondary antibodies.
Whole serum, which contains multiple proteins, can be used as a blocking agent at a 10% concentration. The serum is usually derived from horse or fetal calf. Serum is more expensive than milk or BSA and is less commonly used as a blocking agent.
Whole serum contains immunoglobulins that can potentially cross-react with primary or secondary antibodies leading to high, non-specific background.
Mahmood, Tahrin, and Ping-Chang Yang. "Western blot: technique, theory, and trouble shooting."North American journal of medical sciences4.9 (2012): 429.