High-Throughput Protein–Protein Interactions Screening Using Pool-Based Liquid Yeast Two-Hybrid Pipeline

Because of its adaptability to high-throughput approaches and a low operating cost, the yeast two-hybrid (Y2H) assay remains the most widely used one for high-throughput protein–protein interactions (PPI) mapping experiments. Here we provide a detailed protocol for a liquid culture-based high-throughput binary protein–protein Y2H screen pipeline of a pool of 50 proteins used as baits against a collection of ~12,000 Arabidopsis proteins encoded by sequence-verified open reading frames (ORFs).