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Curr Protoc Plant Biol. 2018 Sep;3(3):e20067. doi: 10.1002/cppb.20067. Epub 2018 Jun 26.

High-Quality Yeast-2-Hybrid Interaction Network Mapping.

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Helmholtz Zentrum München, Institute of Network Biology (INET), Munich, Germany.
Ludwig-Maximilians-Universität München, Microbe-Host Interactions, Munich, Germany.


In this article, we describe a Y2H interaction mapping protocol for systematically screening medium-to-large collections of open reading frames (ORFs) against each other. The protocol is well suited for analysis of focused networks, for modules of interest, assembling genome-scale interactome maps, and investigating host-microbe interactions. The four-step high-throughput protocol has a demonstrated low false-discovery rate that is essential for producing meaningful network maps. Following the assembly of yeast expression clones from an existing ORFeome collection, we describe in detail the four-step procedure that begins with the primary screen using minipools, followed by secondary verification of primary positives, identification of candidate interaction pairs by sequencing, and a final verification step using fresh inoculants. In addition to this core protocol, aspects of network mapping and quality control will be discussed briefly.


high-throughput; interactome; network; protein-protein interaction; systems biology

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