| Assay Method Information | |
| | Binding Assay |
| Description: | The GABAAα5/β3/γ2 protein used for the Scintillation Proximity Assay was derived from membranes produced from HEK293 GABAA α5/β3/γ2 expressing HEK293 cell line. HEK293 GABAA α5/β3/γ2 expressing HEK293 cell line was cultured, then harvested and centrifuged the cells. Membranes were produced by homogenization of the cell pellet with a tissue homogenizer. The resulting homogenate was centrifuged at 48,000 g for 30 minutes, re-suspended and washed twice with 10 mM potassium phosphate buffer, pH 7.4. Final resuspension of membranes took place in buffer containing 10 mM potassium phosphate and 100 mM KCl.For assay, test compound (50×) was added 0.5 μl per well to a white-walled clear-bottomed 384-well plate.Full signal controls were prepared by the addition of 0.5 μl of DMSO to the appropriate wells. Mixture of 10 μl of protein and 10 μl of PVT-WGA beads (0.2 mg/well) were added to the plate. Both protein and bead were diluted to the desired concentration with 10 mM potassium phosphate, pH 7.4, containing 100 mM KCl. The plate was pre-incubated for 30 minutes at room temperature with shaking before initiation of the reaction by the addition of radioligand. 5 μl of [3H]-Ro15-1788 was added at a concentration of 30 nM (5×) and the plates sealed. The reaction mix was incubated at room temperature overnight with gentle agitation on a plate shaker. The final DMSO concentration for all phases of the screening was 2% (v/v). At the end of the overnight incubation, the plates were centrifuged for two minutes at 1,000 rpm prior to reading on a Perkin Elmer Microbeta. |
| Affinity data for this assay | |
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