| Assay Method Information | |
| | Competitive Radioligand Binding Assay |
| Description: | Receptor binding affinities (Ki) were determined from a competitive radioligand binding assay with human recombinant ([125I]-Tyr4)-Angiotensin-II (2200 Ci/mmol) from Perkin Elmer (Cat. #NEX1050). The assays were performed with a scintillation proximity assay (SPA) method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (Perkin Elmer Cat. #RPNQ0001). Assay buffer containing BSA (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% w/v fatty-acid free BSA) was used for preparing radioligand, membrane and SPA bead reagent dilution to working stock concentrations. Reference control and test compounds were diluted to obtain ten-point concentration response curves using a four-fold serial dilution protocol onto assay plates, dispensed acoustically from DMSO stock using automated ECHO instrument technology (Labcyte, Inc.). Concentration response curves were routinely generated with the highest final assay concentration for the reference angiotensin-II control at 50 nM, and the highest final assay concentration for the test compounds at 50 μM. To quantitate the concentration of [125I]-tyr4 angiotensin-II used in each assay, after this radioligand was diluted on the day of testing (2.5-fold working stock), direct counts of this stock were measured by removing four aliquots of 20 μL and counted on a Wizard2 Gamma Counter (PerkinElmer). The hAT2R membrane was combined with PVT-WGA SPA beads to obtain a final assay concentration of 0.25 μg/well hAT2R membrane+0.1 mg/well PVT-WGA SPA bead. And the hAT1R membrane PVT-WGA SPA bead final concentrations was 0.5 μg/well hAT1R membrane+0.1 mg/well PVT-WGA SPA bead. |
| Affinity data for this assay | |
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