Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy.

Mark Soave, Joëlle Goulding, Robert Markus, Stephen J Hill, Leigh A Stoddart

Methods in molecular biology (Clifton, N.J.) 2020

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Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

Citation

Mark Soave, Joëlle Goulding, Robert Markus, Stephen J Hill, Leigh A Stoddart. Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy. Methods in molecular biology (Clifton, N.J.). 2020;2041:163-181