Lab Tools: Close Encounters

Protein-protein interaction assays for all occasions.

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Proteins almost never act alone. In a molecular version of "guilt by association," identifying the function of novel proteins often requires pinning down the proteins with which they interact. But yeast two-hybrid assays and coimmunoprecipitation, the two main techniques for generating "interactomes," maps of protein interactions on a proteome-wide scale, can leave gaping holes.

The yeast two-hybrid (Y2H) assay is genetically simple and amenable to genome-scale analyses. It involves two fusion proteins, called "bait" and "prey," coexpressed in yeast; each is coupled to one half of a transcriptional activator such that their association tethers a transcriptional activation domain to DNA, inducing expression of a reporter gene. The technique has some restrictions, however: The fusion proteins must be overexpressed and localized in the nucleus, and can not include membrane proteins, transcriptional activators, mammalian post-translational modifications, or multiprotein complexes.

Fishing proteins out of cell lysates via coimmunoprecipitation (coIP) and analyzing them using ...

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  • Jeffrey M. Perkel

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