| Assay Method Information | |
| | The Inhibitory Effect of the Compounds on PDE3A |
| Description: | A PDE3A fluorescence polarization (FP) assay in a multi-step format was performed using black, round bottom 384-well plates (Corning, 4514). The testing compounds were serially diluted from stock solutions to 11 concentrations with a 3-fold dilution factor using DMSO. Fifty nl of compound dilutions was mixed with 5 μl of reaction buffer (10 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3, 0.1% phosphate-free BSA) containing 0.2 μM FAM-cAMP (BPS, 60200) and 2 nM PDE3A (Sino Biological, 11908-H20B1) enzyme, and incubated for 60 min at 25° C. Afterwards, 15 μl binding agent mixture (Molecular Devices, R8124) was added to each well and the plate was incubated for 60 min at 25° C. The plate was loaded to a BMG PHERAstar FSX to read Fluorescence Polarization (FP) values at a setting of: Ex: 485 nm and Em: 520 nm. Emission light intensity with polarizers parallel (Emi) and emission light intensity with polarizers perpendicular (Emi) were recorded. The Polarization (mP) values were calculated using the following formula: mP=(SignalEml−SignalEm⊥)/(SignalEml+SignalEm⊥). |
| Affinity data for this assay | |
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