Summary: Immunoblotting (also called Western Blotting) is a technique to separate and identify individual proteins in a protein mixture (e.g. a cell lysate). In Immunoblotting (Western blotting) the protein mixture is applied to a gel electrophoresis (SDS-PAGE gel electrophoresis) to sort the proteins by its molecular weight size in individual protein bands. The separated protein bands are then transferred to a nitrocellulose transfer membrane. The proteins adhere to the membrane in the same pattern as they have been separated due to interactions of charges. To visualize the protein of interest the membrane is commonly first probed using a primary protein- specific antibody followed by a labeled secondary antibody. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. The bound antibodies are then detected by western blotting substrate (e.g. Pierce ECL Western Blotting Substrate), and then visualized by using adeveloping film (HyBlot CL Autoradiography Film) in the darkroom. The thickness of the band corresponds to the amount of protein present.