Immunoprecipitation
Encyclopedia : I : IM : IMM : Immunoprecipitation
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 identify protein complexes present in cell extracts, by targeting a protein believed to be in the complex. The complexes are brought out of solution by insoluble antibody-binding proteins isolated initially from bacteria, such as Protein A and Protein G. These can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex.
Immunoprecipitation is very useful in chromatin immunoprecipitation, which allows analysis of DNA that binds a particular protein.
Steps
- 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 (usually poly-clonal against protein of interest) and allow antibody-antigen complex formation.
- Precipitate the complex of interest using insoluble antibody proteins such as Protein A.
- Analyze complexes or antigens of interest. This can be done in a variety of ways:
- # SDS-PAGE electrophoresis (expose the film if you used radioactivity).
- # Transfer and western blot using another anitbody for proteins that were interacting with the antigen.
- # Other molecular methods.
External links
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