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Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
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Biologia
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JoVE Journal Biologia
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

DOI:
10.3791/1698-v

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14:04 min

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February 01, 2010

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, , ,
1Department of Biochemistry,University of Toronto, 2Department of Molecular Genetics,University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR),University of Toronto

Capítulos

  • 00:00Título
  • 01:22Introduction
  • 01:41Selection of Bait and Appropriate MYTH System
  • 03:04Generation of tMYTH Baits by Gap Repair
  • 03:07a. Vector Preparation and Amplification of Gene of Interest
  • 05:30b. Yeast Transformation and Recovery of Bait Plasmid DNA
  • 07:03Bait Validation
  • 08:10MYTH Screening
  • 08:12a. Large Scale Yeast Transformation
  • 11:31b. Bait Dependency Test
  • 12:54Representative Results/ Outcome
  • 13:12Conclusion

Summary

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MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Tags

Split-ubiquitinMembrane Yeast Two-hybridProtein-protein InteractionsMYTH SystemFull-length Transmembrane ProteinsSaccharomyces CerevisiaeBait ProteinPrey ProteinsUbiquitin MoietiesArtificial Transcription FactorPseudo-ubiquitin MoleculeDeubiquitinating EnzymesReporter Gene Expression

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