| Assay Method Information | |
| | Effect of Compounds on PDE4D2 Activity Assay |
| Description: | The effect of compounds on PDE4D2 activity was detected using a fluorescence polarization kit (BPS Bioscience, Catalog #60345). During the reaction, 12.5 μL of 0.272 ng/well of an enzyme (final concentration: 0.068 ng/well) and 12.5 μL of the compound diluted in a gradient manner (DMSO concentration: 4%) were first pre-incubated at room temperature for 15 minutes, the same volume of the enzyme at the same concentration and 12.5 μL of PDE buffer containing 4% DMSO were added to the positive control well, and 25 μL of PDE buffer containing 2% DMSO was added to the negative control well. After completion, 25 μL of 0.2 M FAM-Cyclic-3′,5′-AMP (final concentration: 0.1 μM) was added to each well, fully mixed, and then incubated at room temperature with slow shaking for 30 minutes. The Binding Agent was diluted with the Binding Agent Diluent (cAMP) at a ratio of 1:100 for later use, the Binding Agent dilution was taken and added to all the wells of the test plate at 100 μl/well and incubated at room temperature with slow shaking for 1 hour. After the incubation was complete, BMG LRBTECH microplate reader was used for FP detection, with Excitation at 485 nm and Emission at 520 nm. |
| Affinity data for this assay | |
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