| Assay Method Information | |
| | Beta-Arrestin2 Recruitment Assay |
| Description: | Agonist-promoted β-arrestin2 recruitment to the sphingosine-1-phosphate 1 receptor was determined using the β-arrestin PathHunter ) Detection kit (DiscoverX Corporation, Cat #: 93-0001). In this system, β-arrestin2 is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor or EA) and the C-terminus of the GPCR of interest is fused to a smaller (42 amino acids), weakly-complementing fragment 15 termed ProLink . In cells that stably express these fusion proteins, stimulation with a cognate agonist results in the interaction of 0-arrestin2 and the Prolink -tagged GPCR. This allows the complementation of the two β-galactosidase fragments and results in the formation of a functional enzyme with β-galactosidase activity. AssayComplete Cell Plating 11 Reagent was removed and replaced with Ham's F-12 containing IBMX (500 μM), and NKH-477 (1.5 μM) along with test or control compounds at the desired concentrations. Following a 60 minute incubation at 37 C. and 5% CO2 in a humidified CO2 and temperature-controlled incubator, the components of the β-arrestin PathHunter Detection kit were added as per the manufacturer's instructions. After an hour incubation at room temperature, plates were analyzed by a BMG PheraStar microplate reader. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |