Array-based yeast two-hybrid (Y2H) screening in combination with a pooling strategy permits identification of complete interaction networks. After discussing the general advantages and drawbacks of Y2H, this chapter provides a detailed protocol for an array-based Gal4p Y2H screen using a pooling strategy to improve efficacy of the experimental work. This includes bait transformation, bait autoactivation testing, pooling of the baits and preys, yeast mating and evaluation of the results. Moreover, a one-on-one confirmation protocol and a quantitative α-galactosidase assay protocol to compare interaction strengths are provided.
Keywords: Arrays; Pooling; Protein–protein interactions; Yeast two-hybrid; α-Galactosidase assay.