| Assay Method Information | |
| | Radioligand binding assay |
| Description: | Radioligand binding assay. Human CGRP receptors expressed (consisting of CRLR and RAMP1) in insect Sf21 cell membrane homogenates were re-suspended in the binding buffer (10 mM HEPES, pH 7.4, 5 mM MgCl2, 0.2% BSA) to a final assay concentration of 0.6 μg protein per well. Saturation isotherms were determined by the addition of various concentrations of 3H-telcagepant (Ho et al, The Lancet, 2008, 372, 2115) (in a total reaction volume of 250 μL) for 60 min at rt. At the end of the incubation, membranes were filtered onto a unifilter, a 96-well white microplate with bonded GF/B filter pre-incubated with 0.5% PEI, with a Tomtec cell harvester and washed 5 times with distilled water. Non-specific binding (NSB) was measured in the presence of 10 nM MK-3207 hydrochloride (CAS No. 957116-20-0). Radioactivity on the filter was counted (1 min) on a microbeta counter after addition of 50 μL of scintillation fluid. For inhibition experiments, membranes were incubated with 0.5 nM 3H-telcagepant and 10 concentrations of the inhibitory compound (0.001-10 μM). IC50 values were derived from the inhibition curve and the affinity constant (Ki) values were calculated using the Cheng-Prussoff equation (Cheng et al, Biochem. Pharmacol. 1973, 22, 3099-3108). |
| Affinity data for this assay | |
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