Western blotting (WB) is an antibody-based method enabling sensitive detection of a protein of interest from other proteins present in biological samples. Detection is made after an initial separation step of the proteins according to their molecular weight, by PolyAcrylamide Gel Electrophoresis (PAGE). After immobilization onto a nitrocellulose, nylon or PVDF membrane, the protein of interest is revealed by its interaction with appropriate primary and secondary antibodies, followed by enzymatic (or fluorescent) methods.
Here are 2 tactics, that we’ve all been taught to keep in mind, but which will really help you towards designing successful WB protocols. A big thank you to Dana and Camillo (Rockland Immunochemicals) for their support here.