Chapter title |
Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.
|
---|---|
Chapter number | 27 |
Book title |
Protein-Protein Interactions
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2425-7_27 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2424-0, 978-1-4939-2425-7
|
Authors |
Wagemans, Jeroen, Lavigne, Rob, Jeroen Wagemans, Rob Lavigne |
Abstract |
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. |
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