0.1M cobalt chloride is added dropwise to saturated calcium hydroxide. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab-Sepharose. 0.2M ferric nitrate is added dropwise to 1M sodium hydroxide. Figure 1 displays the equipment used in the E030 lab with its primary functions. It requires some lab equipment and more advanced chemicals (see item list below). The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. To assess the influence of pH on the phosphate removal process. The Precipitate Lab is perfect for an introduction to chemical reactions or as a review. Immunoprecipitation is achieved with polyclonal anti-immunoglobulin (Ig) serum, anti-Ig-Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Preparation of Ab-Sepharose is described in the Support Protocol. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. The dissociated antigen is then analyzed by electrophoretic methods. Immunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane-bound binding of a specific antigen to an antibody precipitating the antibody-antigen complex washing the precipitate and dissociating the antigen from the immune complex.
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