Live Cell Biosensor Assay Used To Interrogate GPCRs - Comparative Analysis of Biosensor Variants
Application Note Nov 19, 2013
BioTek Instruments Inc.
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 GloSensorTM 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 firefly 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 affinity for cAMP, where the basal signal for this construct can be difficult to detect under select conditions.
This application note compares the performance of the highest and lowest affinity variants using automated methods and the highly-sensitive bottom reading capabilities of an HTS microplate reader.