08_IMMUNODETECTION_110709 - IMMUNODETECTION OF WESTERN...

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IMMUNODETECTION OF WESTERN BLOTTED ANTIGENS LABORATORY EXERCISE #8 Rev: 3/10/10 Materials and Methods Western Blotting (Immunoblotting) was  used to analyze whole-cell lysates and extracellular protein production for staphylococcal enterotoxin. Bacterial proteins were resolved using PAGE and electroblotted to nitrocellulose membranes (Protran BA85; Schleicher & Schull, Keene NH) for 18 hr (TE Series Transphor Electrophoresis Unit; Hoefer Scientific Instruments, San Francisco CA). The membranes were blocked in assay buffer (TBS containing 1% skimmed milk), probed at 37 o C for 90 min with rabbit antisera specific for the target protein (………, diluted 1:1000 in assay buffer), incubated with conjugate (Protein A-HRP; Zymed Laboratories, S. San Francisco CA), then developed with a chromatogenic substrate ( HRP color developing reagent  prepared according to the manufacturer’s instructions; Biorad, Hercules CA).   After developing the blot was washed with TBS and allowed to dry at room temperature. Immunoblotting (often referred to as western blotting ) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. There are three steps in the process: 1. ELECRTOPHORESIS 2. BLOTTING 3. IMMUNODETECTION - The third stage involves " staining " the membrane to visualize the transferred proteins so that they can be identified both by their electrophoretic migration pattern and their antigenic characteristics - The membrane is blocked to prevent nonspecific binding of antibody and then probed with an antibody specific for the antigen(s) of interest (the 1 st Ab) - Because an antigen/antibody complex cannot be seen on the membrane, the 1 st Ab must be labeled with some type of visual marker or "tag" - The tag in turn must be a molecule which can bind to an antibody molecule without destroying the antibody's specific binding ability - Chromogenic enzymes (enzymes which bring about a color change in a colorless substrate) are usually used for the Western blot - The 1 st Ab is next detected by a horseradish peroxidase (HRP)-antiimmunoglobulin (Ig) conjugate* (the 2 nd Ab) and
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