Analysis of Protein–Protein Interaction by Co-IP in Human Cells

Abstract

While there are various approaches available to analyze protein–protein interactions, coimmunoprecipitation (co-IP) remains one of the most classic and commonly used methods to discover novel protein interactions or to determine the physical association of proteins. The assay begins with the preparation of total cell or tissue lysate in an appropriate lysis buffer. Protein of interest in the lysate is captured using a specific antibody and precipitated along with its binding proteins using a resin. After a series of washes to remove nonbound proteins in the lysate, the resultant immune complexes are subjected to immunoblotting, in-gel protein staining, or mass spectrometry to determine the protein–protein interaction of interest. In this chapter, a standard IP/co-IP protocol is described and potential problems and troubleshooting are discussed.