Assay Method Information |
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| Enzyme Inhibition Selectivity Assay |
Description: | Human PDE1A, 3A, 4D2, 5A1, 7B, 8A1, 9A2, and 11A4 enzymes were purchased from BPS Bioscience. Human PDE6AB enzyme was purchased from Scottish Biomedical. Human PDE2A3 was generated from Sf9 transfected with the full-length gene in house. PDE activities were measured using a SPA (Scintillation Proximity Assay) (PerkinElmer). To evaluate the inhibitory activity, 10 μL of serial diluted compounds were incubated with 20 μL of PDE enzyme in assay buffer (50 mM HEPES-NaOH, 8.3 mM MgCl2, 1.7 mM EGTA, 0.1% BSA (pH 7.4)) for 30 min. at room temperature. Final concentration of DMSO in the assay was 1% as compounds were tested in duplicate in 96-well half-area plates (Corning). To start the reaction, 10 μL of substrate [3H] cGMP (PerkinElmer) for PDE1A, 2A3, 5A1, 6AB, 9A2, and 11A4 or [3H] cAMP (PerkinElmer) for PDE3A, 4D2, 7B, and 8A1 was added for a final assay volume of 40 μL. After 60 min incubation at room temperature, yttrium SPA beads containing zinc sulphate were added (20 μL at 6 mg/mL) to terminate the PDE reaction. After being settled for 60 min., assay plates were counted in a scintillation counter (PerkinElmer) to allow calculation of inhibition rate. IC50 values were calculated on the basis of 0% control wells with DMSO and 100% control wells without enzyme. |
Affinity data for this assay | |
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