An alternative to immunofluorescence (see Section A-10) for detecting a protein in tissue sections is immunohistochemistry, in which the specific antibody is chemically coupled to an enzyme that converts a colorless substrate into a colored reaction product in situ. The localized deposition of the colored product where antibody has bound can be directly observed under a light microscope. The antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. This method of detecting bound antibody is analogous to ELISA (see Section A-4) and frequently uses the same coupled enzymes, the difference in detection being primarily that in immunohistochemistry the colored products are insoluble and precipitate at the site where they are formed. Horseradish peroxidase and alkaline phosphatase are the two enzymes most commonly used in these applications. Horseradish peroxidase oxidizes the substrate diaminobenzidine to produce a brown precipitate, while alkaline phosphatase can produce red or blue dyes depending on the substrates used; a common substrate is 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium (BCIP/NBT), which gives rise to a dark blue or purple stain. As with immunofluorescence, the native structure of the protein being sought usually needs to be preserved so that it will be recognized by the antibody. Tissues are fixed by the most gentle chemical fixation techniques, or frozen tissue sections are used that are fixed only after the antibody reaction has been performed.
A-13 Immunoprecipitation and co-immunoprecipitation.
To raise antibodies against membrane proteins and other cellular structures that are difficult to purify, mice are often immunized with whole cells or crude cell extracts. Antibodies against the individual molecules are then obtained by using these immunized mice to produce hybridomas making monoclonal antibodies (see Section A-7) that bind to the cell type used for immunization. To characterize the molecules identified by the antibodies, cells of the same type are labeled with radioisotopes and dissolved in nonionic detergents that disrupt cell membranes but do not interfere with antigen–antibody interactions. This allows the labeled protein to be isolated by binding to the antibody in a reaction known as immunoprecipitation. The antibody is usually attached to a solid support, such as the beads that are used in affinity chromatography (see Section A-3), or to Protein A, a protein derived from the cell wall of Staphylococcus aureus that binds tightly to the Fc region of IgG antibodies. Cells can be labeled in two main ways for immunoprecipitation analysis. All the proteins in a cell can be labeled metabolically by growing the cell in a medium containing radioactive amino acids that are then incorporated into cellular proteins (Fig. A.13). Alternatively, one can label only the cell-surface proteins by radioiodination under conditions that prevent iodine from crossing the plasma membrane and labeling proteins inside the cell, or by a reaction that labels only membrane proteins with biotin, a small molecule that is detected readily by labeled avidin, a protein found in egg whites that binds biotin with very high affinity.
Once the labeled proteins have been isolated by the antibody, they can be characterized in several ways. The most common is polyacrylamide gel electrophoresis (PAGE) of the proteins after they have been dissociated from antibody in the strong ionic detergent sodium dodecyl sulfate (SDS), a technique