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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. StoddartEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)

Abstract

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.

Key words

Fluorescence Antagonist Purinergic receptor BRET Confocal microscopy Ligand binding 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Mark Soave
    • 1
    • 2
  • Joëlle Goulding
    • 1
    • 2
  • Robert Markus
    • 3
  • Stephen J. Hill
    • 1
    • 2
  • Leigh A. Stoddart
    • 1
    • 2
    Email author
  1. 1.Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life SciencesUniversity of NottinghamNottinghamUK
  2. 2.Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
  3. 3.School of Life Sciences Imaging (SLIM), School of Life SciencesUniversity of NottinghamNottinghamUK

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