Document Detail

Time-resolved fluorescence ligand binding for G protein-coupled receptors.
MedLine Citation:
PMID:  23764938     Owner:  NLM     Status:  In-Data-Review    
G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.
Alexander Emami-Nemini; Thomas Roux; Marion Leblay; Emmanuel Bourrier; Laurent Lamarque; Eric Trinquet; Martin J Lohse
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Publication Detail:
Type:  Journal Article     Date:  2013-06-13
Journal Detail:
Title:  Nature protocols     Volume:  8     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-06-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  1307-20     Citation Subset:  IM    
1] Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany. [2] Rudolf Virchow Center, Deutsche Forschungsgemeinschaft (DFG) Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
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