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After Western blot transfer is completed, the membrane undergoes a series of incubation and wash steps to prepare the membrane and probe it for your target protein. The first step, called blocking, coats the membrane surface with a protein or other molecule that is non-reactive to the antibody that will be used to probe for the target protein, or antigen. Nitrocellulose and PVDF membranes have a high protein-binding affinity, which is essential for successful Western blot analysis. The blocking step ensures that the antibody binds specifically to the antigen rather than non-specifically to the membrane surface, which can result in a high background signal that prevents detection of your protein of interest. The membrane is blocked with a buffer that is often comprised of Tris- or phosphate-buffered saline with Tween-20 detergent and up to 5% of a non-reactive protein such as bovine serum albumin, non-fat milk, gelatin or normal serum. The exact formulation and incu
