Application of BRET to monitor ligand binding to GPCRs
2015; Nature Portfolio; Volume: 12; Issue: 7 Linguagem: Inglês
10.1038/nmeth.3398
ISSN1548-7105
AutoresLeigh A. Stoddart, Elizabeth K. M. Johnstone, Amanda J. Wheal, Joëlle Goulding, Matthew B. Robers, Thomas Machleidt, Keith V. Wood, Stephen J. Hill, Kevin D. G. Pfleger,
Tópico(s)Photoreceptor and optogenetics research
ResumoBioluminescence resonance energy transfer, from the substrate of a luciferase fused with a G protein–coupled receptor to a fluorescent dye covalently linked to a receptor ligand, allows the profiling of ligand affinity and binding kinetics. Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.
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