Protocol to characterize Gi/o and Gs protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements

Development of Ligands for the Super Conserved Orphan G Protein-Coupled Receptor GPR27 with Improved Efficacy and Potency

“…We started with two different tests on different cell types to measure the levels of intracellular cAMP that reflect coupling to G s -or G i/o -protein families. The first assay is an end point measurement of cAMP accumulation after 30 min stimulation with the compounds 27 (Figure 9A), while the other is a kinetic measurement based on the cAMP Glo sensor technology in live cells 28 (Figure 9B). These experiments revealed that 7p, 7ab, or 7af did not significantly influence the levels of cAMP through GPR27 (Figure 9A,B).…”

“…A detailed protocol for transient transfection with Lipofectamine 2000 (Thermo Fisher Scientific) and determination of cAMP content of cell extracts using a nonradioactive assay based on the ALPHAScreen technology (PerkinElmer LAS) has previously been published. 27 GloSensor. A detailed protocol has been published previously.…”