Document Detail


Time-resolved fluorescence ligand binding for G protein-coupled receptors.
MedLine Citation:
PMID:  23764938     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
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.
Authors:
Alexander Emami-Nemini; Thomas Roux; Marion Leblay; Emmanuel Bourrier; Laurent Lamarque; Eric Trinquet; Martin J Lohse
Related Documents :
20713068 - Metabotropic glutamate receptor 4 novel agonist lsp1-2111 with anxiolytic, but not anti...
22170608 - Cryptochromes mediate rhythmic repression of the glucocorticoid receptor.
8923528 - G-protein activation by metabotropic glutamate receptors reduces spike frequency adapta...
17917118 - Group i metabotropic glutamate receptors: a role in neurodevelopmental disorders?
17453858 - Selective estrogen receptor modulators and risk for coronary heart disease.
3072128 - Angiotensin ii receptor binding and the baroreflex pathway.
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    
Affiliation:
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.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Impaired left ventricular filling in COPD and emphysema: is it the heart or the lungs? The Multi-Eth...
Next Document:  Ligand-based receptor identification on living cells and tissues using TRICEPS.