Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries

Abstract

Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA?protein (Y1H) and protein?protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic ?test tube? and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).