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after Western blot transfer the membrane is ready to be probed for your target protein of interest or the antigen the detection process involves blocking the membrane in probing with primary and secondary antibodies the blocking step is critical it entails blocking the membrane surface with a protein or another molecule that is nonreactive to the antibody that will be used to probe for your target protein or antigen nitrocellulose and PVDF membranes have a high protein binding affinity which is essential for successful protein transfer the blocking step ensures that the antibody binds specifically to the antigen rather than non specifically to the membrane surface nonspecific binding can result in a high background signal which prevents detection of your protein of interest blocking buffers are often comprised of tryst or phosphate buffered saline between twenty detergent and up to five percent of a nonreactive protein such as bovine serum albumin nonfat milk casein or gelatin there ar
