| Assay Method Information | |
| | Binding Assay of Compounds to AT2R |
| Description: | The assay was performed in accordance with the instruction manual of Angiotensin AT2 Receptor Ligand Binding Assay Kit (#C1TT1AT2, Cisbio). First, a compound stock solution (10 mM) was diluted in a gradient at a 5× dilution ratio (including 10 concentrations, each with two replicates). 160 nL of compounds at different concentrations was added to a 384-well plate. 40 μL of 1×TLB was added to each well, and the mixture was shaken at room temperature for 15 minutes. 15 mL centrifuge tube added with 5 mL of 1×TLB was prepared in advance. The frozen labeled cells were thawed in a water bath at 37° C. (for 1 to 2 minutes), and the thawed cells were quickly transferred to the above 15 mL centrifuge tube. The mixture was mixed well and centrifuged at 1,000 g at room temperature for 5 minutes. The supernatant was removed, and the cells were resuspended by adding 2.7 mL 1×TLB. A new 384-well plate was taken, and 10 μL of mixed cells were added into the corresponding wells according to the assay design. 5 μL of 4× compound solution and 5 μL of 4× Tag-lite red fluorescent labeled ligand were added to each well. After incubation at room temperature for 1 hour, data were read using EnVision with an HTRF mode. The excitation light intensities at 665 nM and 615 nM in each well were read respectively, and the ratio was calculated (Ratio=A665nMIB615 nM). The IC50 value was calculated by means of GraphPad Prism8 software, where X represents logarithmic value of compound concentration; and Y represents ratio of A665 nM/B615 nM. |
| Affinity data for this assay | |
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