title: Homogeneous time-resolved fluorescence assay to probe folded G protein-coupled receptors.
abstract: Continued advances in G protein-coupled receptor (GPCR) structural biology and biochemistry depend in part on strategies to stabilize these polytopic membrane proteins in purified systems. New methods to measure properly folded GPCRs are needed to facilitate the identification of suitable conditions and ensure sample quality. Most GPCRs do not contain an intrinsic reporter on their functionality, so probes must be introduced. Here, we describe a fluorescence-based approach to quantitatively measure the chemokine receptor CCR5 with labeled antibodies. The assay is exceptionally sensitive and high-throughput. We detail procedures to label antibodies, characterize the system, and process data. We also describe several useful applications, including optimization of incorporation into nanoscale apolipoprotein bound bilayers (NABBs or nanodiscs), measurement of receptor stability, and competition binding assays.
authors: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York, USA.
authors: Adam M
authors: AM
authors: Knepp
authors: Thomas P
authors: TP
authors: Sakmar
authors: Thomas
authors: T
authors: Huber
mesh_terms: D001053:Apolipoproteins / Q000737:chemistry; D001667:Binding, Competitive; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D008051:Lipid Bilayers / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
chemical_list: D001053:Apolipoproteins; D007074:Immunoglobulin G; D008051:Lipid Bilayers; D009942:Organometallic Compounds; D019713:Receptors, CCR5; D012996:Solutions; C070734:europium(III) trisbipyridine cryptate
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