MCHr1 antagonist 2

Kinase experiment:

In 96-well plates, IMR-32 cells (I3.4.2) membranes (6 μg/well) are incubated in the presence of test compound (MCHr1 antagonist 2) in binding buffer (25 mM HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2 and 0.5% BSA) and with 0.05 nM [125I]MCH (2200 Ci/mmol) per well for 60 min at room temperature. Nonspecific binding controls consist of I3.4.2 membranes, 0.05 nM [125I]MCH, and 300 nM human MCH. Total binding controls of I3.4.2 membranes and 0.05 nM [125I]MCH are also included on each plate. The plates are centrifuged for 5 min at 1380 g in a GS-6R desktop centrifuge. The reaction buffer is carefully aspirated from each well without disturbing the pellet. Wash buffer (25 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5 M NaCl) is added to each well and then transferred to a 0.5% polyethylenimine-treated GF/B filtration plate using a plate Filtermate Harvester. The filter plate is washed three times with wash buffer, MicroScint 20 is added to each well, and the plate is read using a Topcount microplate scintillation counter[1].

References:

[1]. Lynch JK, et al. Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: assessment of potency, efficacy, and cardiovascular safety. J Med Chem. 2006 Nov 2;49(22):6569-84.