Most proteins need to interact physically with other proteins to be able to perform their biological functions; the identification of new protein-binding partners for a protein of interest may therefore be a key strategy in defining the mechanism of action for a particular protein. Many different approaches to studying protein-protein interactions exist, but identifying which interactions are biologically significant is not trivial. Thus, once an interaction is found, the first step is to validate the suggested interactions using genetic, biochemical, and cell-biological approaches. Here we will focus on three complementary protein interaction approaches: (a) the yeast two-hybrid (YTH) system, a genetic assay performed in living yeast cells, which can be used both to identify proteins that bind to a protein of interest and to determine domains or residues critical for an interaction; and two biochemical approaches, (b) the coimmunoprecipitation of interacting proteins with a specific antibody; and (c) the far-Western method that can show direct physical interaction of two proteins.
KeywordsActivation Domain ADH1 Promoter Bait Plasmid GAL4 Activation Domain Prey Plasmid
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