Western+Blot+(Immunoblot)

__**Western Blot (Immunoblot) **__


 * Basic Description: **

The Western blot, also known as immunoblotting, is a technique first coined by Towbin et al. in 1979 used to identify specific proteins of interest from a mixture; it was named in geographical accordance with Southern blotting, a DNA probing technique.[1] Western blot is a procedure applicable following SDS-PAGE – another protocol used to separate a biological mixture into bands on a polyacrylamide gel by size.[2]


 * Purpose of Technique: **

Such a membrane is capable of binding strongly to proteins even after the transfer has been performed, so the unoccupied binding sites must be blocked a blocking reagent (casein is commonly used) before further steps are taken.[4] Subsequently, a primary monoclonal antibody solution specific to the researchers’ protein of interest can be applied to the solution, and the antibodies will bind to their target. After several wash steps to remove excess antibody, the membrane is then exposed to a secondary polyclonal antibody solution, this time specific only to the primary antibody. The secondary antibody also has an enzyme attached to its end (commonly horse radish peroxidase).[4,5] After several additional washes, the membrane is finally placed in a substrate solution that will cause color change wherever secondary antibody was bound to the membrane.[5]


 * Origin and History: **

Prior to the development of the Western blot, samples run on a gel, while separated, could not be easily probed for additional testing due to the samples being embedded into the gel.[3] This technique makes the sample accessible to probes and other such analyses by electrically transferring the proteins which have been negatively charged with SDS to a nitrocellulose membrane.[2]


 * Recent Research: **

While the Western blot is not the most modern of protocols, it is still a widely used biochemical technique even today. Kouguchi et al. applied this procedure to study Echinococcus multilocularis, a parasite transmitted through dogs that can be lethal to humans. They found that the gene product of the hsp20 gene was commonly seen throughout the organism’s lifecycle using a 2D Western blot (which simply includes isoelectric focusing for better separation), which may provide a target for a new vaccine against this organism.[6]

Pengsuk et al. has also used the Western in their studies. Vibrio cholerae, the microorganism responsible for causing cholera, is currently identified using PCR. However, despite the high sensitivity of PCR, the procedure requires expensive instrumentation as well as skilled technicians, which may be impractical or unavailable in the field. Therefore, the researchers have developed a simpler method to recognize a variety of cholera strains via Fast-Dot blotting. The authors successfully produced and tested seven monoclonal antibodies for cholera identification applicable for dot-blotting through western blot.[7]

A study by Nockler et al. even showed that the Western blot technique was more sensitive than the ELISA when examining pig sera for anti-Trichinella-IgG(98.1 and 96.8% respectively), and was also more specific (100 and 97.9%).[8] Clearly, while the Western blot is by no means a new technique, it is still an extremely useful procedure used by many biologists and biochemists today.


 * References: **

Towbin H., Staehelin T., Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Biochemistry 76: 4350–4354.

Voet, D., Voet, J. G., & Pratt, C. W. (2008). Fundamentals of biochemistry. (3rd ed.). Danvers, MA: Wiley.

Kurien, B.T., Scofield, R.H. (2002). Protein blotting: a review. Journal of Immunological Methods 274: 1-15.

Key Steps for Successful Immunodetection Using the SNAP i.d.TM Protein Detection System. (2008).

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Alberts B., Johnson A., Lewis, J., Raff M., Roberts K., Walter P. (2008) Molecular Biology of the Cell. (5th ed.) New York, NY: Garland Science.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Kouguchi H., Matsumoto J., Katoh Y., Suzuki T., Oku Y., Yagi K. (2008) Echinococcus multilocularis: Two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs. Experimental Parasitology 124: 238–243.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Pengsuk C., Longyant S., Rukpratanporn S., Sridulyakul P., Sithigorngul P. (2011) Differentiation among the Vibrio cholerae serotypes O1, O139, O141 and non-O1, non-O139, non-O141 using specific monoclonal antibodies with dot blotting. Journal of Microbiological Methods 87: 224-233.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Nockler K., Reckinger S., Brogila A., Mayer-Scholl A., Bahn P. (2009) Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera. Veterinary Parasitology 163: 341-347.