| Assay Method Information | |
| | EP3 Binding Activity Measurement |
| Description: | To each well of a 96 well plate, 10 μL of a medium (dimethyl sulfoxide; DMSO) which had been 10-fold diluted with an assay buffer solution (10 mmol/L KH2PO4 KOH containing 1 mmol/L EDTA, 10 mmol/L Mg2+ and 100 mmol/L NaCl, pH6.0) or a DMSO solution of a test compound (final concentration of DMSO: 0.5%), 90 μL of assay buffer solution, 50 μL of 10 nmol/L [3H]-PGE2 (final concentration: 2.5 nmol/L), and 50 μL of human EP3 receptor expressing cell membrane fraction (manufactured by Millipore) (membrane protein mass: 2.5 μg) were placed, and subjected to incubation at room temperature. In nonspecific binding group, instead of the medium, 2 mmol/L of PGE2 was added (final concentration of PGE2: 10 μmol/L). After 60 minutes, a membrane fraction was subjected to suction filtration using a cell harvester, and collected onto a glass fiber (GF/B) plate (hereinafter, filter plate ) which had been wetted with a washing buffer solution (10 mmol/L KH2PO4 KOH containing 100 mmol/L NaCl, pH6.0) in advance. Furthermore, an operation of adding about 0.2 mL of the washing buffer solution to the 96 well plate after suction filtration, and carrying out suction filtration was repeated twice, and the remaining membrane fractions were collected. The filter plate was washed with 150 mL of washing buffer solution twice, and then dried at 50° C. to 60° C. for about 60 minutes. After an accessary back seal was attached to the bottom surface of the filter plate, about 50 μL per well of liquid scintillation cocktail was added to the filter plate, and a scaling film sheet was attached to the upper surface of the filter plate. The filter plate was shaken, and then radioactivity (cpm) of the filter plate was measured using a microplate scintillation counter. A specific binding amount of [3H]-PGE2 to EP3 receptor was calculated by subtracting the radioactivity of the nonspecific binding group from the radioactivity other than the radioactivity of the nonspecific binding group. The inhibition rate by the test compound was calculated from the specific binding amount of [3H]-PGE2 in a medium group and the compound of the present invention group, a Ki value (dissociation constant of the test compound) was calculated from estimated IC50 value (the concentration of the test compound required for inhibiting 50% with respect to the specific binding amount of the medium group) according to the following formula. |
| Affinity data for this assay | |
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