| Assay Method Information | |
| | GABAA Receptor Binding Ability Assay |
| Description: | 1.2.2 35S-TBPS Binding Assay1. 230 μL of PBS was added to each well of a well plate with 1.1 mL volume.2. 60 μL of the cerebral cortex cell membrane (5 μg/μL) solution was added to each well, and the mixture was mixed well.3. The test compound (3 μL per well) was added, and the plate was incubated at 25° C. for 5 minutes. The DMSO concentration was 1%. The initial compound concentration was 1 M, and a3-fold dilution in gradient was carried out to obtain a total of 8 gradients and 2 replicates. 1% DMSO was used as a negative control, and 10 μM P026-2 was used as a positive control.4. GABA was added at a final concentration of 5 μM, and incubated at 25° C. for 5 minutes. 1 mM GABA solution was formulated, and 1.5 μL of the solution was added to each well.5. 35S-TBPS was added at a final concentration of 2 nM. The concentration of isotope mother solution was 9.7 μM. After dilution with PBS for 100 times, 6 μL of the diluted isotope solution was added to each well.6. The plate was incubated at 4° C. for 20 hours.7. The FilterMate GF/C plate was pre-treated with 0.5% PEI, and incubated at 4° C. for 1 hour.8. The FilterMate GF/C plate was washed with Universal Harvester twice, 50 mL PBS each time.9. The reaction solution was transferred to the GF/C plate, and each well was washed 4 times with 900 μL of PBS.10. The washed GF/C plate was placed at 55° C. and dried for 10 minutes.11. 40 μL of scintillation solution was added to each well, and the CPM value was read with TopCount. |
| Affinity data for this assay | |
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