Western Blotting

Western Blotting or immunoblotting (Western blotting was dubbed because it is similar to Southern blotting which was invented by and named after the inventor E. M. Southern) 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.