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Identification of Protein-Protein Interactions by Standard Gal4p-Based Yeast Two-Hybrid Screening

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Protein-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1278))

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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Acknowledgements

Jeroen Wagemans holds a predoctoral fellowship of the “Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen” (IWT, Belgium).

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Authors and Affiliations

  1. Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, 2462, 3001, Leuven, Belgium

    Jeroen Wagemans & Rob Lavigne

Authors
  1. Jeroen Wagemans
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  2. Rob Lavigne
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Corresponding author

Correspondence to Rob Lavigne .

Editor information

Editors and Affiliations

  1. Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA

    Cheryl L. Meyerkord

  2. Department of Pharmacology, Department of Hematology & Medical Oncology, and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, USA

    Haian Fu

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Wagemans, J., Lavigne, R. (2015). Identification of Protein-Protein Interactions by Standard Gal4p-Based Yeast Two-Hybrid Screening. In: Meyerkord, C., Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 1278. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2425-7_27

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  • DOI: https://doi.org/10.1007/978-1-4939-2425-7_27

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2424-0

  • Online ISBN: 978-1-4939-2425-7

  • eBook Packages: Springer Protocols

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