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WESTERN BLOTTING - IMMUNOBLOTTING LABORATORY EXERCISE #7 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/1% skim milk), probed at 37 o C for 90 min with rabbit antisera specific for specific to the target protein (……….), incubated with conjugate (Protein A-HRP; ………), then developed with ……..substrate. Western Blot (alternately, Immunoblot ) is the most commonly used procedure for the detection of a specific protein in a given sample of tissue homogenate or extract. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. (Detection of RNA is termed northern blotting.) - Used extensively in research laboratories for: - Determination of antigen characteristics - Detection of antigens that are difficult to label or precipitate - Purification of antibodies - Assay of the presence, quantity, and specificity of antibodies from different sera - Useful in clinical diagnosis for detection of specific antibody in a patient serum (e.g. testing for antibodies against the agent of Lyme disease ) or proteins of specific disease agents in clinical specimens (e.g. definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease') and detection of the AIDS virus*, since any of the HIV structural proteins can be found in a clinical sample) * The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Commercially, proteins from known HIV-infected cells are separated and blotted on a membrane. Then in the clinical lab, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. 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 - Samples are solubilized , usually with SDS, urea, and reducing agents such as 2-mercaptoethanol - Following solubilization, the material is separated by SDS-PAGE , although thin-layer chromatography can also be used - The process involves gel electrophoresis of the protein mixture to resolve each 1
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component as a separate band (similar to the immunoelectrophoresis technique) 2. BLOTTING - The antigens are then electrophoretically transferred from their posi- tions in the gel to a nitrocellulose membrane , where they are bound irreversibly
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