Screening arrayed libraries with DNA and protein baits to identify interacting proteins

Abstract

Molecular interactions are an integral part of the regulatory mechanisms controlling gene expression. The yeast one- and two-hybrid systems (Y1H/Y2H) have been widely used by many laboratories to detect DNA–protein (Y1H) and protein–protein interactions (Y2H). The development of efficient cloning systems have promoted the generation of large open reading frame (ORF) clone collections (libraries) for several organisms. Functional analyses of such large collections require the establishment of adequate protocols. Here, we describe a simple straightforward procedure for high-throughput screenings of arrayed libraries with DNA or protein baits that can be carried out by one person with minimal labor and not requiring robotics. The protocol can also be scaled up or down and is compatible with several library formats. Procedures to make yeast stocks for long-term storage (tube and microplate formats) are also provided.