Detection of protein-protein interactions by coprecipitation.
Coprecipitation of proteins from whole-cell extracts is a valuable approach to test for physical interactions between proteins of interest. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The approaches described can be adapted for other systems. In a typical experiment, as described here, cells are lysed and a whole-cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid-phase affinity matrix, and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high affinity to a molecule that can be coupled to a solid-phase matrix.
Elion
Elaine A
EA
Harvard Medical School, Boston, Massachusetts, USA.
D000818:Animals; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins