Recursos Técnicos - Posters Científicos
A Cost-Effective Workflow for High-Throughput Screening of G Protein-Coupled Receptors (GPCR)Descargar
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October 28, 2009
Authors: Dee Shen, Joanne Schultz and Wayne F. Patton, Enzo Life Sciences, Farmingdale, NY ; Paul Held and Peter Banks, BioTek Instruments, Winooski, VT
Drugs targeting members of the GPCR super-family represent the core of modern medicine, accounting for the majority of the bestselling drugs and roughly 40% of all prescription pharmaceuticals on the market today. Cell-based assays that monitor the functional activation of GPCRs are thus considered a critical part of the drug discovery process. We describe a new fl uorescent probe for drug discovery which is provided to cells as a cell membrane-permeable acetoxymethyl (AM) ester. Once within the cells, the probe is hydrolyzed by intracellular esterases, leading to the generation of a cell membrane impermeable form, in an analogous manner as commonly used Fluo-3 or Fluo-4 dyes. Intracellular calcium binding to the fluorescent probe is readily measured using a fl uorescence microplate reader equipped with a dual-reagent dispenser. This fl uorescence-based assay workflow is suitable for monitoring calcium mobilization across a broad spectrum of biological targets. The easyto- use protocol is automation-friendly and can be performed in a convenient 96-well or 384-well microplate format. The described assay workfl ow results in low sample-to-sample variability and excellent Z’ values in a miniaturized format. The ease-of-use of the kit, affordability and high performance of the instrument and minimal installation requirements make the described workfl ow readily accessible, even to academic research groups with modest consumables and instrumentation budgets.