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Transcript
What is a yeast two hybrid assay? How has this approach been used to look globally or genome-wide at protein-protein interactions? What is a one hybrid assay? Jamie Feldmann Shweta Lahudkar John Martin Yeast two hybrid assay z z z z Performed in S. cerevisiae most commonly Binding Domain Activating Domain Interact without direct binding Yeast two hybrid assay z z z z z Reporter transcript Known protein as bait cDNA library If interact, reporter transcript activated Positive vs Negative Selection z Summary of process How has this approach been used to look globally or genome-wide at protein-protein interactions Yeast two hybrid assay z z z Functions of new found proteins inferred through interactions with known proteins Expected interactions Æ what reacts with known proteins By mutating amino acids, can determine importance of those remaining zFig. 2: General flowchart of a hypothetic two-hybrid screen. Pointed lines should be followed in case of negative results z z Yeast Two-Hybrid: State of the Art Wim Van Criekinge1* and Rudi Beyaert2 1 DEVGEN NV. Technologiepark 9, 9052 Zwijnaarde-Gent. Belgium. Phone: 32-9-264-5403 Fax: 32-9-264-54-18 2 Department of Molecular Biology. Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000, Ghent. Belgium. What is a one hybrid assay? One hybrid assay z z z Used for protein-DNA interactions AD is directly bound to BD BD not of fixed sequence, may be a library Disadvantages z z z z z Known sequence for AD or BD Need a protein of known function Have to assume each cell only takes up one plasmid Fusion might change shape of bait protein Interpreting significance Advantages z z z z Can be done in vivo One plasmid is sufficient Only need indirect interaction Yeast is host Thank You!!