Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding

British Journal of Pharmacology
Leigh A StoddartK D G Pfleger

Abstract

Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd ) and competition (Ki ) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel bioluminescence r...Continue Reading

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Methods Mentioned

BETA
BRET
saturation binding
competition binding
Fluorescence
confocal microscopy
confocal
flow cytometry
flow
Fluorescence resonance
FRET

Software Mentioned

BRET
GraphPad Prism

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