07ip_Commentary n References_112108

07ip_Commentary n References_112108 - Immunoblotting...

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COMMENTARY Background Information Immunoblotting is a rapid and sensitive assay for the detection and characterization of proteins. It can be used to identify particular proteins by exploiting the specificity inherent in antigen-antibody recognition. Immunoblotting is extremely powerful, as it combines electrophoretic separation of proteins, glycoproteins, and lipopolysaccharides with immunological identification. Immunoprecipitation has been widely used to visualize the antigens recognized by various antibodies, both polyclonal and monoclonal. However, there are several problems inherent with immunoprecipitation, including (1) the requirement for radiolabeling of antigen, (2) co-precipitation of tightly associated macromolecules, (3) occasional difficulty in obtaining precipitating antibodies, and (4) insolubility of various antigens (Talbot et al., 1984). To circumvent these problems of immunoprecipitation, electroblotting (Towbin et al., 1979) - subsequently popularized as “western blotting” or immunoblotting (Burnette, 1981) - was conceived. This procedure involves the solubilization and electrophoretic separation of macromolecules by SDS-PAGE or urea-PAGE followed by quantitative transfer and irreversible binding to either nitrocellulose or diazobenzyl-oxymethyl (DBM) paper. This technique has been useful in identifying specific antigens recognized by poly- clonal or monoclonal antibodies, and it is highly sensitive (i.e., 1 η g of antigen can often be detected). Protein blotting has important clinical applications-it is the confirmatory test for human immunodeficiency virus Type 1 (HIV1). SDS-PAGE-separated virus proteins are blotted onto nitrocellulose and processed with patient sera. After washing and incubating (using a Tween 20/nonfatdry milk diluting and washing solution) with an anti-human IgG coupled to HRP or alkaline phosphatase, the antigens are identified by chromogenic development. Typically, the prepared blots and all reagents needed for the test are purchased commercially (Bio-Rad Novapath; Organon Teknika Cappel HIV-l Western Blot Kit; Du PontlBiotech HIV Western Blot Kit). Critical Parameters First and foremost, the antibody should recognize denatured antigen. Nonspecific binding of antibodies can occur, so control antigens and antibodies should always be run in parallel. The time of transfer and the first antibody and conjugate dilutions should always be optimized. A variety of agents are currently used to block binding sites on the membrane after blotting (Harlow and Lane, 1988). These include Tween 20, PVP, nonfat dry milk, casein, BSA, and serum. O.l % (v/v) solution of Tween 20 in TBS (TTBS), a convenient alternative to protein-based blocking agents, is recommended for chromogenic development of nitrocellulose and PVDF membranes (Blake et aI., 1984). In contrast to dry milk, TBS blocking solution (BLOTTO), TTBS is stable and has a long shelf life at 4°C. Furthermore, TTBS generally produces a clean background and permits subsequent
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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07ip_Commentary n References_112108 - Immunoblotting...

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