| Assay Method Information | |
| | Measurement of the Inhibitory Action of the Compound of the Present Invention on Binding of [33P]-S1P to S1P5 (EDG-8) |
| Description: | Compound of the Present Invention on Binding of [33P]-S1P to S1P5 (EDG-8)A reaction was carried out in a 96-well microplate by using membrane fractions of Chinese hamster ovary (CHO) cells each of which was made to overexpress human S1P1 (EDG-1) or human S1P5 gene respectively, in an amount of the membrane fraction of 1 mg protein/mL. To each of the wells, 100 μL of a vehicle (DMSO) solution or a two-fold concentration-ligand solution each of which was diluted with Binding Buffer (50 mmol/L, Tris pH 7.5, 5 mmol/L, MgCl2, 0.5% BSA and Complete EDTA free (1 tablet/50 mL)), and 50 μL of 0.16 nmol/L [33P]-S1P (manufactured by American Radiolabeled Chemicals, Inc.) diluted with Binding Buffer were added. Thereafter, the membrane fraction solutions (50 μL) was added to the wells and the reaction was carried out at room temperature for 60 minutes. After the reaction, suction filtration was carried out by using a 96-well UNIFILTER, and the 96-well microplate was washed with Wash Buffer (50 mmol/L, Tris pH 7.5, 0.5% BSA) (150 mL), and thereafter, was dried at 60° C. for 45 minutes. MicroScint (trade name) 20 (50 μL/well) was added and the plate was covered with TopSeal-A, and thereafter, the radioactivity was measured by using TopCount (manufactured by PerkinElmer Inc.). |
| Affinity data for this assay | |
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