NanoBRET<sup>TM</sup> assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar® and the PHERAstar® platereaders. — ASN Events
  1. Schedule
  2. Poster Session 2
  3. NanoBRETTM assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar® and the PHERAstar® platereaders.

NanoBRETTM assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar® and the PHERAstar® platereaders. (#136)

John Abbenante 1 , Leigh A Stoddart 2 , Elizabeth K. M. Johnstone 3 , Amanda J Wheal 2 , Joëlle Goulding 2 , Matthew B Robers 4 , Thomas Machleidt 4 , Keith V Wood 4 , Stephen J Hill 2 , Kevin D. G. Pfleger 3
  1. BMG Labtech, Mornington, VIC, Australia
  2. Cell Signalling Research Group, School of Life Sciences, The University of Nottingham Medical School, Nottingham, United Kingdom
  3. Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, Nedlands, WA, Australia
  4. Promega Corporation, Madison, Wisconsin, USA

The study of ligand binding to GPCRs and the monitoring of cellular responses to these external stimuli remains a prime focus for medical research. This paper describes a highly sensitive BRET (Bioluminescence Resonance Energy Transfer) assay (NanoBRET) that allows the monitoring of ligand binding to GPCRs in live cells. The method relies on expression of a newly developed, low molecular weight, Nanoluc luciferase on the N terminus of a GPCR in live cell lines. Monitoring of ligand binding and competitive inhibition studies were efficiently achieved by utilising a fluorescently labelled agonist of the receptor. Ligand binding data and competition assays for the b2 adrenergic receptor will be presented.

 

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  3. Stoddart, L. et al. Nat. Methods 12, 661-663 (2015)
  4. Baker, J.G. Br. J. Pharmacol. 144, 317–322 (2005)