Immunoprecipitation

The immunoprecipitation technique has the advantage of allowing the isolation of the antigen of interest for further analysis. It also provides a sensitive assay for the presence of a particular antigen in a given cell or tissue type. An extract produced by disruption of cells or tissues is mixed with an antibody against the antigen of interest in order to form an antigen-antibody complex that will precipitate. However, if the antigen concentration is low (often the case in cell and tissue extracts), the assembly of antigen-antibody complexes into precipitates can take hours, even days, and it is difficult to isolate the small amount of immunoprecipitate that forms.

Fortunately, there are a number of ways to avoid these limitations. One is to attach the antibody to a solid support, such as a synthetic bead, which allows the antigen-antibody complex to be collected by centrifugation. Another is to add a secondary antibody specific for the primary antibody to bind the antigen-antibody complexes. If the secondary antibody is attached to a bead, the immune complexes can be collected by centrifugation. A particularly ingenious version of this procedure involves the coupling of the secondary antibody to magnetic beads. After the secondary antibody binds to the primary antibody, immunoprecipitates are collected by placing a magnet against the side of the tube (Figure 6-13).

When used in conjunction with biosynthetic radioisotope labeling, immunoprecipitation can also be used to determine whether a particular antigen is actually synthesized by a cell or tissue. Radiolabeling of proteins synthesized by cells of interest can be done by growing the cells in cell-culture medium containing one or more radiolabeled amino acids. Generally, the amino acids used for this application are those most resistant to metabolic modification, such as leucine, cysteine, or methionine. After growth in the radioactive medium, the cells are lysed and subjected to a primary antibody specific for the antigen of interest. The Ag-Ab complex is collected by immunoprecipitation, washed free of unincorporated radiolabeled amino acid and other impurities, and then analyzed. The complex can be counted in a scintillation counter to obtain a quantitative determination of the amount of the protein synthesized. Further analysis often involves disruption of the complex, usually by use of SDS and heat, so that the identity of the immunoprecipitated antigen can be confirmed by checking that its molecular weight is that expected for the antigen of interest. This is done by separation of the disrupted complex by SDS-PAGE and subsequent autoradiography to determine the position of the radiolabeled antigen on the gel.

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Essentials of Human Physiology

Essentials of Human Physiology

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