Protein Protocols Protein Protocols

Immunoprecipitation 1097
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Immunoprecipitation
Kari Johansen and Lennart Svensson
1. Introduction
Immunoprecipitation allows the investigator to detect and quantitate antigens in a
mixture of proteins or characterize a specific antibody response to already well-characterized
proteins. Addition of antibodies to proteins, usually radiolabeled, allows formation
of antigen–antibody complexes. After separation from contaminating proteins,
the complexes are disassociated and the proteins of interest are separated by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Size and quantity
of proteins may then be analyzed either by autoradiography or a gel scanning procedure.
Immunoprecipitation is extremely sensitive and may detect very small amounts
of radiolabeled protein (detection level ~100 pg protein or 100 cpm/protein). Unlabeled
proteins may be used if other sensitive detection methods are utilized, e.g., enzymatic
activity assays or Western blotting. The advantage of the immunoprecipitation
technique vs immunoblotting is the possibility to analyze the immune response to proteins
expressed in their native conformation. Radioimmunoprecipitation assay (RIPA)
is used routinely for the detection of viral proteins, characterization of monoclonal and
polyclonal antibody preparations, and determination of the specificity of the immune
response to various pathogens (1–3).
The major steps in immunoprecipitation are:
1. Labeling of proteins expressed by mammalian cells;
2. Lysis of the cells;
3. Addition of antibodies to lysed cells and formation of antigen–antibody complexes;
4. Purification of the specific immune complexes; and
5. Analysis of the immunoprecipitated proteins.
1.1. Metabolic Labeling of Proteins
Several techniques are available for labeling proteins. Usually, radiolabeled essential
amino acids, such as 35S-labeled methionine and 35S-labeled cysteine, are used to
label newly synthesized proteins expressed in mammalian cells, either naturally or after
transfection or infection. 35S emits a weak β-radiation easily detected either by autoradiography
or gel scanning and has a half-life of 87.1 d. Although most strains of yeasts
and prokaryotes synthesize methionine and cysteine (1), 35S-labeled sulphate is used as
the primary metabolic precursor to label proteins in these organisms.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
1097