Western Blotting - A technique to find proteins
The western blot or protein immunoblot or western blotting, is an analytical technique to detect specific proteins in molecular biology and immunogenetics from a sample of tissue extract.
Specific protein complex mixture can be accomplished by a technique western blotting named besides techniques like southern blotting and northern blotting. Each of these technologies has its specific identification process like where southern blotting meant for DNA fragment detection and northern blotting is for mRNAs. Western blotting was identified by Dr. George stark that uses antibodies to locate proteins.
WORKING OF WESTERN BLOTTING:
In this process, a protein mixture is separated through the electrophoretic method on an SDS-polyacrylamide gel (SDS-PAGE). A dissociating agent like sodium dodecyl sulfate (SDS) is used where the gel is infused in it. Through electrophoresis process the protein bands are identified and individual protein bands are detected by flooding the nitrocellulose membrane with a radiolabeled or enzyme-linked polyclonal or monoclonal antibody specific for the protein to be detected. The protein molecules on the membrane could easily bind with the antibody. This works like Ag-Ab binding.
These bounded form of enzyme-linked antibody and the protein of interest could be identified in many different ways. As it is a radioactive labelled antibody molecule, they can be specified and identified by exposing the membrane to a sheet of x-ray film, a procedure known as autoradiography. The detection mostly employs enzyme-linked antibodies against protein. The colored band at the site of the target region is achieved through the addition of a chromogenic substrate. The protein site can be easily determined with a higher sensitivity if a chemiluminescent compound along with suitable enhancing agents which can produce light at the antigen site.
Specific antibodies can also be detected through the western blotting technique in a mixture of antibodies. So well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. This procedure is in confirmatory testing for HIV and even the patient with antibodies that react with one or more viral proteins can be defined. Analysis of western blotting is carried out using different methodologies like luminescence, color reaction, etc.
But, it is a time-consuming process and also have a high demand in terms of the experience of the experimenter also it requires optimizing the experimental conditions it includes of protein isolation, buffers, type of separation, gel concentration.