Place a piece of blotting paper onto the sponge support pad.Place a sponge support pad onto the cassette and remove the air bubbles by gently rolling a Pasteur pipette over the pad.Submerge the open Transfer Cassette cathode plate onto the tray pre-filled with Western Transfer Buffer.Set up the Submerged Western Transfer Cassette as described below and in Figure III-2:.(The electrophoresis can be followed by using pre-stained molecular weight markers). Load the protein sample onto a 4-20% Tris-Glycine polyacrylamide gel and run until desired resolution is achieved.Commercially available alkaline phosphatase conjugate substrate kit.Secondary antibody (Donkey-anti-probing antibody species conjugated to Alkaline Phosphatase).Antigen specific probing antibody (Primary Antibody).Methanol.200mL (20% final volume)ĭiH2O.add to 1.0L final volume Tris Base.1.45g (12mM final concentration) *Western Transfer Buffer (1/2 x TOWBIN).(Note: The membrane should be handled with gloves and clean forceps to avoid contamination with extraneous proteins.) A nitrocellulose membrane, approximately the size of the gel, presoaked in Western Transfer Buffer* for five minutes.Submerged Western Transfer Cassette and accessories.Typical Western Transfer and Development Protocol The membrane containing the protein bands is serially incubated with: a suitable blocking reagent to prevent non-specific protein binding, a wash solution to rinse any unbound blocking reagent, a probing antibody (anti-protein-X antibody) that forms a specific immune complex with Protein-X, additional wash solution to remove any unbound antibody, an enzyme-linked antibody that binds specifically to the Fc region of the anti-Protein-X antibody, additional wash solution to remove any unbound enzyme-linked antibody, and finally, a substrate solution, which in the presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rendering the Protein X band visible (Figure III-1). for the detection of protein X) includes the following steps: The proteins in the tested solution are separated into distinct bands by SDS-PAGE, The size-separated proteins are transferred from the polyacrylamide gel to a nitrocellulose membrane (or another suitable matrix). The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, and the ability of immunochemical reagents to interact specifically with a given protein antigen.Ī typical Western-transfer protocol (e.g. Western Transfer, also known as Western Blotting, is a rapid immunoblotting technique for identifying the presence of a particular protein in a complex mixture of proteins such as cell lysates or sera.

Western blot