| Assay Method Information | |
| | Receptor Binding Test |
| Description: | 1. Experimental Materials:(1) D2 receptor Cell Transfection:This experiment utilizes the plasmid vector containing the gene of D2 receptor protein for transfecting HEK293 cells by calcium phosphate transfection method. By culturing the transfected cells in culture medium containing G418 and selecting cell clones followed by radioligand binding test, a stable cell strain capable of expressing the D2 receptor protein stably is finally obtained.(2) Materials for the Receptor Binding Test:Isotopic ligand [3H] Spiperone (113.0 Ci/mmol) purchased from Sigma Corporation; (+) spiperone purchased from RBI Corporation; GF/B glassfiber filter purchased from Whatman Corporation; imported and subpackaged Tris; PPO and POPOP purchased from Shanghai No. 1 Reagent Factory; and fat-soluble scintillation fluid. Beckman LS-6500 multifunction liquid scintillation counter.2. Experimental Methods: (1) Cells:HEK-293 cells are infected with the recombinant viruses containing various genes. 48-72 h later, receptor proteins are massively expressed on the membrane. The cells are centrifuged at 1000 rpm for 5 min, then the culture supernatant is discarded and the cell pellets are collected, preserved in refrigerator at −20° C., and resuspended with Tris-HCl reaction buffer (pH=7.5) before use.(2) Competitive Receptor Binding Test:The compound to be tested, the radioligand (20 μL for each) and the receptor protein (160 μL) are added to a reaction tube, wherein the compound to be tested and the positive drug both have the final concentration of 10 μmol/L.After being incubated in water bath at 30° C. for 50 min, the tube is immediately transferred to ice bath to stop the reaction. The mixture is subjected to rapid suction filtration through GF/C glassfiber filter on Millipore cell sample collector, washed with eluent (50 mM Tris-HCl, pH 7.5) (3 mL×3) and dried under microwave for 5-6 min. The filter is transferred to a centrifuge tube (0.5 mL), added with 500 μL fat-soluble scintillation fluid, left in the dark for over min, and counted to determine the radioactivity intensity. The percentage inhibition ratio for each compound to inhibit the isotopic ligand binding is calculated with the following equation:inhibition ratio (1%)=(total binding tube CPM−compound CPM)/(total binding tube CPM−non-specific binding tube CPM)×100%Each compound is tested in duplicate, and the tests are carried out independently twice.Compounds with an inhibition ratio of over 95% are subjected to the receptor binding test at a series of concentrations so as to determine half maximal inhibitory concentration (IC50, the concentration of the compound required for inhibiting 50% binding of [3H]-Spiperone to D2 receptor). Each concentration is tested in duplicate, and tests are carried out independently twice for each compound. |
| Affinity data for this assay | |
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