Assay Method Information

Assay Name:  Measurement of Binding Affinity for Serotonin 5-HT7 Receptor
Description:  Human gene recombinant 5-HT7 receptor expressed in Chinese hamster ovary (CHO) cells was used. [3H]LSD 1 nM, the 5-HT7 receptor membrane (15 μg/well), each of the samples of Example 1-20 at various concentrations, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM EDTA were added to a vessel until the final volume reached 0.25 ml. The reaction mixture was incubated at 25 C. for 90 min. The incubated reaction mixture was rapidly passed through a Whatman GF/C glass fiber filter, which had been previously soaked with 0.3% polyethyleneimine, using a Brandel-Harvester to quench the reaction and washed with cold 50 mM Tris-HCl buffer. The filter was covered with MeltiLex, sealed in a sample bag, and dried in an oven. The radioactivity retained in the filter was counted using MicroBeta (Wallac). Non-specific binding was determined in the presence of 0.5 μM Mianserin. The Ki value of the test drug was calculated from a nonlinear regression (GraphPad Prism Program, San Diego, USA) of the isotherm curves obtained from 2 repeated tests for two test tubes with concentrations of 10th and 11th steps test.
Affinity data for this assay
 

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