title: Type-3 BRET, an improved competition-based bioluminescence resonance energy transfer assay.
abstract: We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom. Electronic address: james.felce@rdm.ox.ac.uk.
authors.forename: James H
authors.initials: JH
authors.lastname: Felce
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Rachel G
authors.initials: RG
authors.lastname: Knox
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
mesh_terms: D000818:Animals; D004735:Energy Transfer; D008163:Luminescent Measurements / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled