Monitoring G Protein Activation in Cells with BRET

Methods Mol Biol. 2015;1335:107-13. doi: 10.1007/978-1-4939-2914-6_8.

Abstract

Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here, we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using a standard laboratory equipment.

MeSH terms

  • Bioluminescence Resonance Energy Transfer Techniques / methods*
  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • HEK293 Cells
  • Humans
  • Indicators and Reagents / chemistry
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Spectrometry, Fluorescence
  • Transfection

Substances

  • Indicators and Reagents
  • GTP-Binding Proteins