Respected Researchers,
I would like to know what is the principle behind the Western blotting? How will be the expression of interest protein if treated cancer cell with drugs? I read many articles related but still cant sort it out.
Thank you.
The basic procedure for western blotting is as follows.
Lysates prepared from cancerous cells treated or not with the drug of interest are mixed with protein sample buffer containing SDS (to aid protein denaturing and give a global negative charge to all proteins in the sample) and reducing agents (e.g. DTT or beta-mercaptoethanol, to reduce disulphide bonds). Proteins are denatured by heat and loaded onto polyacrilamide gels, where after a electrical current is applied, proteins will migrate towards the positive pole at the end of the gel (because of SDS negative charge). This allows protein fractionation based on their molecular masses: smaller proteins locate at the bottom of the gel whilst larger proteins remain closer to the top. Following, proteins fractionated in the gel are transfered, again with the use of electrical current, to a membrane (typically, nitrocellulose), where the proteins will be immobilized exactly in the same position they were in the gel.
Then, the membrane is incubated with a protein-rich suspension (like BSA or skimmed milk) in a buffered solution (like PBS) to block non-specific binding during the following steps. A primary antibody the detects the target protein is added, and then a secondary antibody that detects the primary antibody (e.g. anti-IgG of the species where the primary antibody was raised). The secondary antibody is conjugated to a reporter, usually horseradish peroxidase, which activity is detected by colorimetric or chemiluminescent assays. The signal intensidy depends on the levels of expression of the protein of interest recognized by the primary antibody. To assess if equivalent protein amounts were loaded onto the gel, one also has to detect the levels of a protein of constitutive expression, like the housekeeping proteins GAPDH or actin.
Good luck!
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