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Real-Time BRET Assays to Measure G Protein/Effector Interactions

  • Darlaine Pétrin
  • Mélanie Robitaille
  • Terence E. HébertEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

Advances in imaging assays based on resonance energy transfer (RET) have made it possible to study protein/protein interactions in living cells under physiological conditions. It is now possible to measure the kinetics of changes in these interactions in response to ligand stimulation in real time. Here we describe protocols for these assays focusing on the basal and ligand-stimulated interaction between tagged Gβγ subunits and adenylyl cyclase II. We describe relevant positive and negative controls and various experimental considerations for optimization of these experiments.

Key words

G protein-coupled receptor Bioluminescence resonance energy transfer G proteins Protein–protein interaction assays 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research to T.E.H (MOP-36279) as well as the CIHR Team in GPCR Allosteric Regulation (CTiGAR). T.E.H. is a Chercheur National of the Fonds de la Recherche en Santé du Québec (FRSQ). We thank Vic Rebois (NIH) for helpful discussions.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Darlaine Pétrin
    • 1
  • Mélanie Robitaille
    • 2
  • Terence E. Hébert
    • 1
    Email author
  1. 1.Department of Pharmacology and TherapeuticsMcGill UniversityMontréalCanada
  2. 2.Leslie Dan Faculty of PharmacyUniversity of TorontoTorontoCanada

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