title: Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.
abstract: Yeast two-hybrid (Y2H) screening permits identification of completely new protein interaction partners for a protein of interest, in addition to confirming binary protein-protein interactions. After discussing the general advantages and drawbacks of Y2H and existing alternatives, this chapter provides a detailed protocol for traditional Gal4p-based Y2H library screens in Saccharomyces cerevisiae AH109. This includes bait transformation, bait auto-activation testing, prey library transformation, Y2H evaluation, and subsequent identification of the prey plasmids. Moreover, a one-on-one mating protocol to confirm interactions between suspected partners is given. Finally, a quantitative α-galactosidase assay protocol to compare interaction strengths is provided.
authors: Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, 2462, 3001, Leuven, Belgium.
authors: Jeroen
authors: J
authors: Wagemans
authors: Rob
authors: R
authors: Lavigne
mesh_terms: D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry; D015723:Gene Library; D005822:Genetic Vectors; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D000519:alpha-Galactosidase / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000519:alpha-Galactosidase