Identifying Interactors from an Activation Domain Prey Library
Program in Systems Biology; Program in Molecular Medicine
Biochemistry, Biophysics, and Structural Biology | Genetics and Genomics | Laboratory and Basic Science Research | Systems Biology
In yeast hybrid assays, the process of identifying preys that interact with the bait of interest involves several steps. First, in this protocol, the bait yeast strain is transformed with a library of activation domain (AD)-prey clones and plated on selective media containing 3-aminotriazole (3AT). This selects transformants containing an AD-prey clone that induces HIS3 reporter expression. Second, these "HIS-positive" colonies are analyzed for LacZ induction (and, optionally, URA3 induction in yeast two-hybrid (Y2H) assays). Third, yeast PCR is used on these "double-positive" colonies to amplify the insert from the AD-prey plasmid. Fourth, some of this PCR product is used to perform a gap-repair retest to confirm the interaction in fresh bait-strain yeast, and the remainder is used for DNA sequencing to determine prey identity for those that successfully retest. Finally, interactions are carefully examined to filter out likely false-positive interactions. This protocol takes 20-43 d plus sequence confirmation to complete.
DOI of Published Version
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):pdb.prot094987. doi: 10.1101/pdb.prot094987. Link to article on publisher's site
Cold Spring Harbor protocols
Reece-Hoyes, John S. and Walhout, Albertha J. M., "Identifying Interactors from an Activation Domain Prey Library" (2018). Program in Systems Biology Publications and Presentations. 142.