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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 four day 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.
Lecture Topics:
Introduction to the yeast two-hybrid system; Yeast as a Genetic Tool; Two-hybrid system components; The Interaction Trap system as an example; Bait Design, Cloning and Assessment; Bait Improvement & Troubleshooting; Library Transformation and Screening; Characterization of primary candidates; Interaction mating and related techniques; Isolation of library plasmids and second confirmation of interaction; False Positives: introduction; Two-hybrid on the Internet; Variations & New Developments (one-hybrid, three-hybrid etc.); Two-Bait systems and Dual Bait system: introduction; Commercially available systems; Bacterial two-hybrid systems; Combined yeast-bacterial system based on Dual Bait; Bioinformatics: protein-protein interaction screening in silico and visualization of protein-protein interactions (Cytoscape)
Laboratory Topics:
Mating assay; Yeast Transformation; Bait Assessment; Replica Technique; b-gal Assay Techniques: on Filters, on Plates and by CHCl3 Overlay; b-gal Assay in Liquid; Yeast DNA Prep and PCR; Advanced Techniques in Mating: Velveteen vs. Replicator; High-throughput b-gal assay in Liquid Using 96-well Plates; Dual Bait : Simultaneous Two-Color Assay; Retrieving and Visualization of Protein-Protein Interactions (Computer Lab)
Course Director, Dr. Ilya Serebriiiskii at the Fox Chase Cancer Center.
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