Two-Hybrid Systems: Methods and Protocols

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Paul N. MacDonald has assembled a collection of powerful molecular tools for examining and characterizing protein protein, protein DNA, and protein RNA interactions. The techniques range from the most basic (introducing plasmids into yeasts, interaction assays, and recovering the plasmids from yeast), to the most advanced alternative strategies (involving one-hybrid, split two-hybrid, three-hybrid, membrane recruitment systems, and mammalian systems). Methods are also provided for dealing with the well-known problem of artifacts and false positives and for identifying the interacting partners in important biological systems, including the SMAD and nuclear receptor pathways. To ensure ready reproducibility and robust results, each technique is described in step-by-step detail by researchers who employ it regularly.