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Genetiske og biokjemiske tilnærminger for<em> I Vivo</em> og<em> In Vitro</em> Vurdering av Protein Oligomerisering: The Ryanodine Receptor Case Study
JoVE Journal
Biologia
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JoVE Journal Biologia
Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Genetiske og biokjemiske tilnærminger for<em> I Vivo</em> og<em> In Vitro</em> Vurdering av Protein Oligomerisering: The Ryanodine Receptor Case Study

DOI:
10.3791/54271-v

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12:43 min

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July 27, 2016

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, ,
1Wales Heart Research Institute,Cardiff University School of Medicine, College of Biomedical and Life Sciences

Capítulos

  • 00:05Título
  • 01:10Y2H: Yeast Transformation
  • 03:22Y2H: Colony-lift Filter Paper β-Gal Assay
  • 04:29Protein Expression in a Mammalian Cell Line
  • 06:56Co-immunoprecipitation
  • 09:02Chemical Cross-linking
  • 09:46Results: The Ryanodine Receptor Forms Homo-oligomers Within Cells
  • 11:40Conclusion

Summary

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Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Tags

Keywords: Protein OligomerizationRyanodine ReceptorYeast Two-hybridCo-immunoprecipitationChemical Cross-linkingProtein-protein InteractionsHomo-oligomersCardiopathiesCardiac ArrhythmiasSudden DeathAnti-arrhythmic DrugsCompetent Yeast CellsPlasmid DNALithium AcetatePEG SolutionDimethyl SulfoxideMinimal SD Medium Plates

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