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Multiplexed GPCR Assays using HTRF® TechnologyDownload
April 29, 2009
Authors: Laurence Jacquemart, Estelle NGarwate, Marie-Laure Lebreton, Cisbio Bioassays; Peter Banks, Xavier Amouretti, BioTek Instruments
GPCRs carry information within cells via two major signaling pathways: regulation of cAMP levels and increases in intracellular Ca2+ triggered by inositol (1,4,5) tri-phosphate (IP3). These signaling pathways are activated by the specific G protein associated with the receptor. Gs- and Gi-coupled receptors regulate cAMP levels while Gq-coupled GPCRs activate phospholipase C (PLC) and trigger the inositol phosphate (IP) cascade. Here we present an HTplex™ assay from Cisbio that allows for quantification of both cAMP levels and activation of the IP cascade in the form of IP1 using HTRF technology.
The IP1 assay is based on the competition between native IP1 produced by cells and IP1 tracer for limited amounts of a Lumi4®-Tb cryptate-labelled monoclonal antibody specific for IP1; the cAMP assays are similarly based on the competion between native cAMP produced by cells and cAMP tracer for limited amounts of a Lumi4®-Tb cryptate-labelled monoclonal antibody specific for cAMP. Differentiation between the two assays is based on using different acceptor dyes on the respective tracers: the cAMP tracer emits in the green portion of the electromagnetic spectrum while the IP1 tracers emit in the red. Data using a model system of stably-transfected vasopressin R2 in CHO cells will be presented.
Synergy™ 2 Multi-Detection Microplate Reader.