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| | PDE Enzyme Inhibition Assay |
| Description: | PDE activity was measured using a plate-based Scintillation Proximity Assay (SPA). For competitive enzyme inhibition assays, the substrate [3H]cAMP concentration was held at 20 nM for conditions to be at or below the Km of the enzyme (24 nM at RT). The concentration of enzyme was adjusted to convert less than 10% of available substrate to end product during the assay. Following the addition of the test compounds and [3H]-cAMP, enzyme was added. The incubation was allowed to proceed for 30 min at room temperature before the addition of PDE SPA beads at 0.2 mg/well to stop the reaction. Plates were allowed to stand 10 to 12 hours before counting in a Trilux plate reader. IC50s were calculated after the subtraction of background as determined by addition of 10 uM papaverine. |
| Affinity data for this assay | |
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