The Protein Man's Blog | A Discussion of Protein Research

Antibodies are used to detect specific proteins during research. There are a variety of tests including the Western blot, that produce a surface area that can contain other proteins and components that need to be blocked. Blocking buffers are used to bind the surface areas that may attach to reagents during the testing process, preventing the non-specific antibodies from interacting with the reagents and causing a poor reading or inaccurate results. Knowing which buffers to use, however, is critical for ensuring the accuracy of a test, as each option has its own advantages and disadvantages.

Immunodetection of proteins on a Western blot requires the use of expensive antibodies, but poor or incomplete transfer would result in wasting these antibodies. A Western transferred blot can be checked for success of protein transfer with a protein stain and therefore prevent wasting expensive antibodies. Various options for protein stains for Western blot are available, including, but not limited to, Ponceau S, Amido black 10B, Coomassie brilliant blue R250, India ink or colloidal gold. In addition to checking success and quality of Western transfer, staining blots is also used for semi-quantitative protein estimation. In addition, checking the blot with a stain gives a rough idea to a researcher that the desired protein is present (based on size, mobility etc) and whether to go for immunodetection especially when expensive and limiting amounts of antibodies are available.

We recently reviewed the advantages of total protein membrane stains as a loading control for Western blotting and demonstrated how they were an improvement on routinely used housekeeping genes.

What's best? Housekeeping genes or total protein for Western blot loading controls

Western blotting is a very popular and sensitive protein detection system that is routinely used for multiple areas of protein research.  The sensitivity of this detection system continues to improve with increasingly sensitive enhanced chemiluminescence systems and the increasing popularity of IR (infra-red) fluorophores and IR imaging systems: