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TRAC 15: Two Hybrid Selection: Identification and Characterization of Protein-Protein Interaction
To characterize a protein’s function, it is often advantageous to identify other proteins with which it interacts. The Yeast Two-Hybrid system is one of the most versatile methods available for detection and characterization of protein–protein interactions, and in the recent years it has become a mature and robust technology. It has successfully been used to detect proteins and peptides that target specific motifs in proteins, to define the domains of amino acids critical for such interactions, and to score specific drug-mediated disruption of protein–protein interaction. It has also been applied to detect interactions between DNA and proteins, RNA and proteins, and between small molecules/drugs and proteins.
This lecture/laboratory course is designed to provide the student with all the information, experience and most of the reagents necessary to design and perform Two Hybrid selection in the laboratory setting. Emphasis will be placed on practical experimental design as well as good theoretical understanding of system’s advantages and limitations.
Topics: Practical recommendations for yeast work; background on Two-Hybrid selection in yeast; "Bait" construction and Assessment; Library screening; Characterization and confirmation of positive candidates; False positives; Overview of YTH modifications and competing systems; Brief overview of Dual Bait system;Yeast transformation; Replica Plating; ß-gal Assays (quantitative and qualitative); Mating Assay; PCR from yeast; DNA isolation from yeast.
Course Director, Dr. Ilya Serebriiiskii at the Fox Chase Cancer Center.
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