Fig. A.13 Cellular proteins reacting with an antibody can be dissociates it from the antibody and also coats the protein with a characterized by immunoprecipitation of labeled cell lysates. strong negative charge, allowing it to migrate according to its size All actively synthesized cellular proteins can be labeled metabolically in polyacrylamide gel electrophoresis (PAGE). The positions of the by incubating cells with radioactive amino acids (shown here for labeled proteins are determined by autoradiography using X-ray methionine; 35S-Met), or one can label just the cell-surface proteins film. This technique of SDS-PAGE can be used to determine the by using radioactive iodine in a form that cannot cross the cell molecular weight and subunit composition of a protein. Patterns of membrane or by a reaction with the small molecule biotin, detected protein bands observed with metabolic labeling are usually more by its reaction with labeled avidin (not shown). Cells are lysed with complex than those revealed by radioiodination, owing to the detergent and individual labeled cell-associated proteins can be presence of precursor forms of the protein (right panel). The mature precipitated with a monoclonal antibody attached to beads. After form of a surface protein can be identified as being the same size as unbound proteins have been washed away, the bound protein that detected by surface iodination or biotinylation (not shown). is eluted in the detergent sodium dodecyl sulfate (SDS), which generally abbreviated as SDSPAGE. SDS binds relatively homogeneously to proteins, conferring a charge that allows the electrophoretic field to drive protein migration through the gel. The rate of migration is controlled mainly by protein size (see Fig. A.13). Proteins of differing charges can be separated using isoelectric focusing (see Section A-7). This technique can be combined with SDS-PAGE in a procedure known as twodimensional gel electrophoresis. For this, the immunoprecipitated protein is eluted in urea, a nonionic solubilizing agent, and run on an isoelectric focusing gel in a narrow tube of polyacrylamide. This first-dimensional isoelectric focusing gel is then placed across the top of an SDS-PAGE slab gel, which is then run vertically to separate the proteins by molecular weight (Fig. A.14). Two-dimensional gel electrophoresis is a powerful technique that allows many hundreds of proteins in a complex mixture to be distinguished from one another.
Immunoprecipitation and the related technique of immunoblotting (see Section A-14) are useful for determining the molecular weight and isoelectric point of a protein as well as its abundance, distribution, and whether, for example, it undergoes changes in molecular weight and isoelectric point as a result of processing within the cell.
Coimmunoprecipitation is an extension of the immunoprecipitation technique and is used to determine whether a given protein interacts physically
Fig. A.14 Two-dimensional gel electrophoresis of MHC class II molecules. Proteins in mouse spleen cells have been labeled metabolically (see Fig. A.13), precipitated with a monoclonal antibody against the mouse MHC class II molecule H2-A, and separated by isoelectric focusing in one direction and SDS-PAGE in a second direction at right angles to the first (hence the term two-dimensional gel electrophoresis). This allows one to distinguish molecules of the same molecular weight on the basis of their charge. The separated proteins are detected by autoradiography. The MHC class II molecules are composed of two chains, α and β, and in the different MHC class II molecules these have different isoelectric points (compare upper and lower panels). The MHC genotype of mice is indicated by lowercase superscripts (k, p). Actin, a common contaminant, is marked a. Photographs courtesy of J.F. Babich.