| Assay Method Information | |
| | Affinity Activities |
| Description: | The affinity of the compound to each subtype of GABAA receptor was determined by competing for the binding of 3H-flunitrazepam to HEK293 cells stably expressing human α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2 receptors. The radioligand competition binding assay was performed in a 200 μL system (96-well plate) containing 100 μL of cell membrane. The concentration of 3H-flunitrazepam was 1 nM and the concentration of the test compound was in the range of 1×10−5-10−6 M. Flumazenil was used as a control. 1 μL of 2 mM flumazenil (final concentration 10 M) was added to the low signal control well (Low control, LC), and 1 μL of DMSO was added to the high signal control well (High control, HC). The final concentration of target membrane protein was 5 g/well. All test compound sample storage solutions were 10 mM. The working concentration of the samples was to dilute all the samples to 0.2 mM with DMSO, followed by 4-fold serial gradient dilutions for a total of 8 concentration gradients. The 96-well plate was sealed with a sealing film, and then incubated on a shaker at room temperature for 1 hour. At the same time, the GF/C filter plate was soaked in soaking buffer (0.3% PEI, stored at 4° C.) for at least 0.5 hours. After the binding incubation was completed, the cells were collected onto a GF/C filter plate using a cell harvester. The plate was washed four times with washing buffer (50 mM Tris-HCl, pH 7.4, stored at 4° C.). After drying in a 50° C. oven for 1 hour, the bottom of the dried GF/C filter plate was sealed. The residual radioactivity on the filter membrane was detected using liquid scintillation counting. 50 μL of scintillation fluid was added to each well, and the plate was sealed. The readings were taken using a Microbeta2. The inhibitory activity of the test sample on the binding of 3H-flunitrazepam to GABAA receptor membrane proteins was calculated, and the IC50 of each test sample was calculated by dose-effect curve fitting (GraphPad Prism 5 software), and the Ki of the sample was calculated from the IC50, thus evaluating the binding ability of the sample to the various subtypes of GABAA receptors. |
| Affinity data for this assay | |
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