Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries
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).
Key wordsArrayed libraries DNA–protein interaction High-throughput One-hybrid system Open reading frame Protein–protein interaction Transcription factors Two-hybrid system Yeast
The work in L.O.-S. lab is supported by MINECO grants BIO2013-46076-R and BIO2016-77840-R. We thank all people that contributed to the development of the Arabidopsis TF library  as well as all the labs that have used this resource helping this way to ascertain its potential. Dr. Begoña Benito is also thanked for critical reading. We apologize to those publications not cited here due to space limitations.
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