AbstractCoprecipitation 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.
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