scholarly journals High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors

2000 ◽  
Vol 5 (2) ◽  
pp. 63-69 ◽  
Author(s):  
Michael Allen ◽  
Julian Reeves ◽  
Geoffrey Mellor
Keyword(s):  
Cell Surface ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Radioligand Binding ◽  
Low Cost ◽  
G Protein Coupled Receptors ◽  
Cell Surface Receptors ◽  
Binding Assays ◽  
Surface Receptors ◽  
G Protein Coupled

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and δ-opioid) and nonpeptide (β-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at β1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.

scholarly journals Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

2014 ◽  
Vol 15 (11) ◽  
pp. 19700-19728 ◽  
Author(s):  
Fabio Cattaneo ◽  
Germano Guerra ◽  
Melania Parisi ◽  
Marta De Marinis ◽  
Domenico Tafuri ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
G Protein Coupled

scholarly journals Fluorescence Polarization Assays for High Throughput Screening of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Rank Order ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Microtiter Plates ◽  
High Throughput Drug Screening ◽  
G Protein Coupled ◽  
Speed Of Analysis

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.

scholarly journals Impact of a Red-Shifted Dye Label for High Throughput Fluorescence Polarization Assays of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (5) ◽  
pp. 329-334 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
False Negative ◽  
G Protein Coupled Receptors ◽  
Negative Results ◽  
False Negative Results ◽  
Assay Precision ◽  
G Protein Coupled

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.

scholarly journals Cell-Free Assay of G-Protein-Coupled Receptors Using Fluorescence Polarization

2008 ◽  
Vol 13 (5) ◽  
pp. 424-429 ◽  
Author(s):  
Jessi Wildeson Jones ◽  
Tiffani A. Greene ◽  
Christine A. Grygon ◽  
Benjamin J. Doranz ◽  
Martha P. Brown
Keyword(s):  
G Protein ◽  
Fluorescence Polarization ◽  
G Protein Coupled Receptors ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Free System ◽  
Molecular Binding ◽  
Soluble Cell ◽  
G Protein Coupled ◽  
Peptide Interaction

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). ( Journal of Biomolecular Screening 2008:424-429)

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