Assay Method Information

Assay Name:  Radioligand Binding Assay
Description:  32 μg membrane proteins of CHO cell expressing human 5-HT6 receptor, 2 nM of radioactive marker [3H]LSD, a compound of the present invention having different test concentrations, 100 μM 5-HT (5-HT was used to eliminate nonspecific binding sites) and a buffer solution were mixed uniformly. Then the resulting mixture was incubated at 37 C. for 120 min, in which the buffer solution comprised 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5 mM EDTA, 10 μM pargyline and 20 mg/L protease inhibitor.After incubation, the resulting mixture was filtered by a fiberglass filter (GF/B, Packard) in vacuo, and the filter membrane of the fiberglass filter was preimpregnated with 0.3% PEI before the filtering and washed with 50 mM of Tris-HCl for several times after the filtering. After the filter membrane was dried, and the radioactivity of filter membrane was determined by liquid scintillation counting by using a scintillometer (Topcount, Packard). The reference standard was 5-HT, and competitive inhibition curves were plotted based on several inhibition ratios and the corresponding compound concentrations. IC50 values were calculated by non-linear regression analysis using Hill equation curves, and the Ki values were calculated from IC50 by using the ChengPrusoff equation.
Affinity data for this assay
 

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