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Automated Processing of a Homogeneous phospho-ERK1/2 Assay for Use as a Generic GPCR Sensor下載
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January 26, 2010
Authors: Paul Held, Peter Banks, BioTek Instruments, Inc., Winooski, VT, USA; Kristin Huwiler, Matthew Robers, Bonnie Hanson, Life Technologies, Madison, WI, USA
Drugs targeting members of the G-protein coupled receptor (GPCR) super-family represent the core of modern medicine, accounting for the majority of the best-selling drugs and roughly 40% of all prescription pharmaceuticals on the market today. Activation of GPCRs can result in stimulation of numerous signaling networks, several of which have been established to result in phosphorylation of Extracellular Regulated Kinase 1/2 (ERK 1/2). As a result, measurement of ERK 1/2 phosphorylation can serve as a surrogate for GPCR activation. We have combined BacMam-mediated gene delivery of a GFP-ERK2 sensor with LanthaScreen® Cellular Assay technology for a time-resolved fluorescence resonance energy transfer (TR-FRET) based measurement of intracellular phospho-ERK2 levels. Together these technologies enable a flexible platform for measuring GPCR activation in the cell line of interest. Here we demonstrate that the assay can be performed in a homogeneous format and adapted to automation. Details regarding workflow, assay performance, and application to GPCR antagonist identification will be illustrated.