| Assay Method Information | |
| | Binding Assay for γ2-Containing GABAA Subtypes |
| Description: | Table 1: Radioligand binding assays were carried out in a volume of 200 uL (96-well plates) which contained 100 uL of cell membranes, [3H]Flumazenil at a concentration of 1 nM and the test compound in the range of [0.1 10+-3+-10] 10+-6 M. Nonspecific binding was defined by 10+-5 M Diazepam and typically represented less than 5% of the total binding. Assays were incubated to equilibrium for 1 hour at 4 C. and harvested onto GF/C uni-filters (Packard) by filtration using a Packard harvester and washing with ice-cold wash buffer (50 mM Tris; pH 7.5). After anhydrousing, filter-retained radioactivity was detected by liquid scintillation counting. Ki values were calculated using Excel-Fit (Microsoft) and are the means of two determinations.The compounds of the accompanying examples were tested in the above described assay, and the preferred compounds were found to possess large Ki value for displacement of [3H]Flumazenil from the alpha1beta3gamma 2 subtype of the human GABAA receptor of 100 nM or above. Most preferred are compounds with a Ki alpha1beta3gamma 2 (nM) >300. In a preferred embodiment the compounds of the invention are binding selectively for the gamma 1 subunit-containing GABAA receptors relative to gamma 2 subunit-containing GABAA receptors. |
| Affinity data for this assay | |
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