| Assay Method Information | |
| | In vitro Radioligand Binding Assay |
| Description: | Equilibrium dissociation constants, Kd, for many of the compounds described in the Examples section (“test compounds”) are determined in the presence cellular membrane prepared from SF9 cell lines overexpressing the human MRGX2 receptor. The assay is carried out in a 96-well plate (Greiner V-Bottom #651201). To each well is admixed fixed amounts of cell membrane preparation (75 μg/well, final concentration) and a 3H-labeled ligand, 3-(((furan-2-yl-4,5-t2)methyl)amino)-5-(o-tolyl)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (Quotient Bioresearch, 40 μM final concentration), along with a range of the (unlabeled) test compound (eight-point dose response curve, 10 μM maximum concentration, 4-fold serial dilution) in 50 mM HEPES buffer containing 10 mM MgCl2, 0.01% Triton X-100, 200 μM EDTA at pH 7.4. The assay mixtures, which comprise MRGX2 receptor, radiolabeled ligand and test compound, are incubated at room temperature (˜22° C.) for 60 minutes to reach equilibrium. After incubation, the assay mixtures are collected on a filter mat (Filtermat A, Perkin Elmer) using a cell harvester (Harvester 96, TOMTEC). The filter mat is completely dried. A solid scintillant (Meltilex, Perkin Elmer) is added to each filter membrane and the level of radioactivity (“Signal”) from each well (assay mixture) is recorded using a scintillation counter (Trilux Microbeta, PerkinElmer). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |