| Assay Method Information | |
| | Radioligand competition binding assay |
| Description: | The assay was carried out in a 200 μL system (96-well plate from Agilent) with 100 μL of cell membrane. The concentration of 3H-flunitazepam (from PerkinElmer, lot: NET567250 UC) was 1 nM and the concentration of test compound was in the range 1×10−5-10−6 M. Flumazenil (from Tocris; lot: 1318) was used as a control. 1 μL of 2 mM flumazenil (final concentration 10 μM) was added to the Low signal control well (Low control, LC) and 1 μL of DMSO (from Sigma aldrich, lot: 472301) was added to the High signal control well (High control, HC). The final target membrane protein concentration was 5 μg/well. All test compound sample stocks were 10 mM DMSO solutions. The working concentration of the samples is to dilute all samples with DMSO to 0.2 mM, and then perform 4-fold continuous gradient dilution for a total of 8 concentration gradients. The 96-well plate was sealed with a plate sealing membrane and then incubated on a shaker at room temperature for 1 hour. Meanwhile, GF/C filter plate was soaked with a plate soaking buffer (0.3% PEI, polyethyleneimine, purchased from Sigma aldrich, model: P314, stored at 4° C.) for at least 0.5 hours. After completion of binding incubation, the cells were collected with a cell collector onto the GF/C filter plate and washed four times with a plate washing buffer (50 mM Tris-HCl, pH 7.4, stored at 4° C.). After drying in an oven at 50° C. for 1 hour, the dried GF/C filter plate was bottom-sealed with a membrane, and the residual radioactivity of the filter membrane was detected by liquid scintillation counting method with 50 μL of scintillation fluid added per well and sealed, and the use of Microbeta2 (Microplate counter, purchased from PerkinElmer, Model: CNLL0153) for reading. The inhibitory activity of the test samples on the binding of 3H-flunitrazepam to the GABAA receptor membrane protein was calculated, the IC50 of each test sample was calculated by dose-effect curve fitting (by GraphPad Prism 5 software), and the Ki (inhibition constant) of the sample was calculated by IC50, thereby evaluating the binding ability of the compounds to BZD sites of α5-GABAA receptor and α2-GABAA receptor. |
| Affinity data for this assay | |
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