Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.

Jeroen Wagemans, Rob Lavigne

Methods in molecular biology (Clifton, N.J.) 2015

filter terms:

Yeast two-hybrid (Y2H) screening permits identification of completely new protein interaction partners for a protein of interest, in addition to confirming binary protein-protein interactions. After discussing the general advantages and drawbacks of Y2H and existing alternatives, this chapter provides a detailed protocol for traditional Gal4p-based Y2H library screens in Saccharomyces cerevisiae AH109. This includes bait transformation, bait auto-activation testing, prey library transformation, Y2H evaluation, and subsequent identification of the prey plasmids. Moreover, a one-on-one mating protocol to confirm interactions between suspected partners is given. Finally, a quantitative α-galactosidase assay protocol to compare interaction strengths is provided.

Citation

Jeroen Wagemans, Rob Lavigne. Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening. Methods in molecular biology (Clifton, N.J.). 2015;1278:409-31