Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1).
In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.