Detection of protein-protein interactions by coprecipitation.

Coprecipitation of proteins from whole-cell extracts is a valuable approach to testing for physical interactions between proteins of interest. When a precipitating antibody is used, this method is referred to as co-immunoprecipitation. Coprecipitation can be used to study interactions between known proteins and as a means of identifying components of a complex. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The extract is prepared under nondenaturing conditions, the protein of interest is precipitated, and the precipitate is tested for 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 bind with high affinity to a molecule that can be coupled to a solid-phase matrix. An associated protein is detected by separating the precipitated proteins by SDS-PAGE and immunoblotting with a second antibody that recognizes the putative associated protein.