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immunoblotting often referred to as western blotting is used to identify within a protein sample specific antigens recognized by polyclonal or monoclonal antibodies this procedure typically follows one or 2d gel electrophoresis and involves the transfer of separated proteins to nitrocellulose membranes incubation of these membranes with primary and secondary antibodies directly or indirectly conjugated to enzymes and visualization of protein antigens on these membranes using colored or fluorescent substrate reactions in this video we provide a step-by-step demonstration of an immunoblotting procedure hi im sean gallagher from uvp i collaborate with a proteomic center here at the keck graduate institute today im going to be showing you immunoblotting analysis it involves a number of steps including protein separation blotting of the proteins onto membranes immunoprobing and visualization with chromogenic and chemoluminescent substrates so lets get started before starting the immunob