Chapter Four - Analysis of Protein–Protein Interactions by Coimmunoprecipitation

Proteins generally act by binding to other molecules, including proteins. When proteins bind to other proteins, we speak of protein–protein interactions. It has become apparent that protein–protein interactions are critically important to many processes that take place in the cell, including signal transduction, regulation of gene expression, vesicular transport, nuclear import and export, and cell migration (Pawson and Nash, 2003). This has led to the recognition of protein–protein interactions as targets for drug development and to an increased interest in the identification of novel protein–protein interactions (Fry and Vassilev, 2005; Fry, 2006 ;  Tord et al., 2007). Coimmunoprecipitation is a technique that is used to confirm novel protein–protein interactions in the context of a living cell or organism. In addition, coimmunoprecipitation is also used to study the dynamics of protein–protein interactions in response to intra- or extracellular stimuli, or can be used to study the effect of mutations on the ability of a protein to engage its binding partner. In a coimmunoprecipitation experiment, a protein of interest is isolated by immunoprecipitation. Subsequently, the presence of binding partners can be assessed by immunoblotting (see Western Blotting using Chemiluminescent Substrates).