Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity. Co-immunoprecipitation (also known as a pull-down) can identify interacting proteins or protein complexes present in cell extracts: by precipitating one protein believed to be in a complex, additional members of the complex are captured as well and can be identified. The protein complexes, once bound to the specific antibody, are removed from the bulk solution by capture with an antibody-binding protein attached to a solid support such as an agarose bead. These antibody-binding proteins (Protein A, Protein G, Protein L) were initially isolated from bacteria and recognize a wide variety of antibodies. Following the initial capture of a protein or protein complex, the solid support is washed several times to remove any proteins not specifically and tightly bound through the antibody. After washing, the precipitated protein(s) are eluted and analyzed using gel electrophoresis, mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex. Thus, co-immunoprecipitation is a standard method to assess protein-protein interaction.
- Lyse cells and prepare sample for immunoprecipitation. (you can radiolabel cells to easily assay for your protein later)
- Pre-clearing the sample using serum from the animal species in which the primary antibody was raised (this decreases background and non-specific proteins).
- Incubate solution with antibody against protein of interest (antibody can be attached to solid support before or after this step) and allow antibody-antigen complex formation.
- Precipitate the complex of interest, removing it from bulk solution.
- Wash precipitated complex several times. Spin each time between washes and remove the supernatant. After the last wash, get rid of as much supernatant as possible.
- Elute proteins from solid support (usually with low-pH or SDS sample loading buffer).
- Analyze complexes or antigens of interest. This can be done in a variety of ways:
- SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); expose the film if you used radioactivity.
- Transfer and Western Blot using another antibody for proteins that were interacting with the antigen.
- Other molecular methods.
- Analysis of Proteins Using Immunoprecipitation at ufl.edu
- Immunoprecipitation at the US National Library of Medicine Medical Subject Headings (MeSH)
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