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.
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D002457:Cell Extracts; D011506:Proteins