Protein blotting is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies. There are different blotting protocols (dot blot, 2D blot); one of the most powerful is western blotting.
Western Blotting or immunoblotting allows investigators to determine, with a specific primary antibody, the relative amounts of the protein present in different samples. Briefly, 1) Samples are prepared from tissues or cells that are homogenized in a buffer that protects the protein of interest from degradation; 2) The sample is separated using SDS-PAGE and then transferred to a membrane for detection; 3) The membrane is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the membrane. A primary antibody is then added to the solution which is able to bind to its specific protein; 4) A secondary antibody-enzyme conjugate, which recognizes the primary antibody is added to find locations where the primary antibody bound.
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