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Live-Cell Biosensor Assay Used To Interrogate GPCRs: Comparative Analysis of Biosensor Variantsダウンロード
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January 14, 2013
Authors: P. Brescia, P. Banks, BioTek Instruments, Inc., Winooski, Vermont; B. Binkowski, P. Stecha, N. Cosby, Promega Corporation, Madison, Wisconsin
Cell Culture Procedure Functional, cell-based assays are essential to identify modulators of GPCRs, including those that signal via increases or decreases in cAMP. Previously, we demonstrated the automation of the GloSensor™ cAMP Assay from Promega, which utilizes a live-cell biosensor consisting of a fusion of a cAMP binding domain to a circularly permuted form of fi refl y luciferase. Upon binding to cAMP, conformational changes in the expressed sensor lead to increases in light output in the presence of substrate. Several variants of the biosensor exist with different affinities for cAMP, some offering increased sensitivity and others offering increased dynamic range. The sensor with the broadest dynamic range, 22F, has the lowest affi nity for cAMP, where the basal signal for this construct can be difficult to detect under select conditions. Here we compare the performance of the highest and lowest affinity variants using automated methods and the highly-sensitive bottom reading capabilities of an HTS microplate reader. Sensitive detection of the 22F basal signal allows for use of this variant under a broad range of conditions, providing an assay with both superior dynamic range and sensitivity.