A stringent yeast two-hybrid matrix screening approach for protein-protein interaction discovery

The yeast two-hybrid (Y2H) system is currently one of the most important techniques for protein–protein interaction (PPI) discovery. Here, we describe a stringent three-step Y2H matrix interaction approach that is suitable for systematic PPI screening on a proteome scale. We start with the identifi cation and elimination of autoactivating strains that would lead to false-positive signals and prevent the identifi cation of interactions. Nonautoactivating strains are used for the primary PPI screen that is carried out in quadruplicate with arrayed preys. Interacting pairs of baits and preys are identifi ed in a pairwise retest step. Only PPI pairs that pass the retest step are regarded as potentially biologically relevant interactions and are considered for further analysis.