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Related Experiment Video

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A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
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Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries.

Rocío Sánchez-Montesino1, Luis Oñate-Sánchez2

  • 1Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|June 18, 2017
PubMed
Summary

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This study presents a simple, mating-based method for high-throughput yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H) screenings. The protocol efficiently detects DNA-protein and protein-protein interactions without robotics, suitable for various library formats.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H) systems are established methods for detecting DNA-protein and protein-protein interactions, respectively.
  • These systems utilize Saccharomyces cerevisiae as a eukaryotic system, making them accessible to many laboratories worldwide.
  • Advancements in cloning have enabled the creation of large open reading frame (ORF) libraries for yeast-based screenings.

Purpose of the Study:

  • To describe a novel, simple, mating-based method for high-throughput screening of yeast one-hybrid and yeast two-hybrid interactions.
  • To provide a protocol that does not require robotic automation for screening arrayed ORF libraries against DNA or protein baits.
  • To offer a scalable and adaptable method compatible with various microtiter plate formats.
Keywords:
Arrayed librariesDNA–protein interactionHigh-throughputOne-hybrid systemOpen reading frameProtein–protein interactionTranscription factorsTwo-hybrid systemYeast

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Main Methods:

  • A mating-based protocol for high-throughput yeast screenings was developed.
  • The method facilitates the screening of arrayed open reading frame (ORF) libraries using DNA (Y1H) or protein (Y2H) baits.
  • The protocol is designed for manual execution, adaptable for different scales, and compatible with 96- and 384-well microtiter plates.

Main Results:

  • The described method allows for high-throughput screening of yeast one-hybrid and yeast two-hybrid interactions.
  • The protocol is efficient, requiring approximately 10 hours of labor spread over 5 days for one person.
  • The system is scalable, from testing few interactions to potential robotization, and supports multiple library formats.

Conclusions:

  • A simple, labor-efficient, and scalable mating-based method for yeast one-hybrid and yeast two-hybrid screenings has been established.
  • This protocol provides an accessible alternative for detecting DNA-protein and protein-protein interactions, particularly for labs without access to robotics.
  • The method's compatibility with various library formats and scalability enhances its utility in diverse research settings.