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Assay Method Information

Assay Name:  Inhibition Assay
Description:  By a method similar to the measurement of PDE9-inhibiting activity, PDE5-inhibiting activity of each of the test compounds was measured, percent inhibition was calculated and IC50 value against PDE5 was determined. To 150 uL of buffer B (70 mmol/L Tris-HCl, pH7.5, 16.7 mmol/L MgCl2, 33.3 nmol/L [3H]-cGMP) solution containing [3H]-cGMP (specific activity=244.2 GBq/mmol) at a concentration of 33.3 nmol/L, 50 uL of a solution of the compound to be evaluated (formed by dissolving the compound in DMSO and diluting it with distilled water to DMSO concentration of 5%) and 50 uL of the PDE9 protein solution as prepared in the above, as diluted with buffer C (40 mmol/L Tris-HCl, pH7.5, 15 mmol/L benzamidine, 15 mmol/L 2-mercaptoethanol, 1 ug/mL Pepstatin A, 1 ug/mL Leupeptin) by 1,500x, were added under cooling with ice. This mixed solution was incubated at 30 C. for 30 minutes and the enzymatic reaction of PDE9 was terminated by heating the system in boiling water.
Affinity data for this assay
 

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Last update November 1, 2007
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