| Assay Method Information | |
| | In Vitro Assay of the Inhibitory Activity of Compounds Against PDE 3A and PDE 4B2 Enzymes |
| Description: | 1) Prepare FAM cAMP working solution: 20 μL of FAM-Cyclic-3′, 5′-AMP stock was added to 1980 μL of PDE assay buffer. The mixture was added to all wells at 25 μL/well.2) Prepare compound solution: the test compounds were dissolved in DMSO to make a 10 mM stock solution. The compound stock solution was diluted in DMSO to a 100× Top Dose diluent. 5 μL of 100× Top Dose diluent was added to 45 μL of PDE assay buffer to prepare a compound Top Dose working solution. Then, the compound working solution was subjected to double dilution with PDE assay buffer containing 10% DMSO to prepare compound working solutions of various concentrations. The compound working solutions were added to compound wells at 5 μL/well. 5 μL of PDE assay buffer containing 10% DMSO was added to each control well.3) Prepare PDE enzyme solution: PDE3A and PDE4B2 enzyme stock solutions were diluted to 150 pg/μL and 50 pg/μL, respectively, with PDE assay buffer, and added to all compound wells and Vehicle control wells at 20 μL/well. 20 μL of PDE assay buffer was added to Blank control wells.4) React at room temperature for 1 h.5) Prepare Binding Agent: 80 μL of binding agent was added to 7920 μL of binding agent diluent, and the mixture was mixed well and added to all wells at 100 μL/well.6) React at room temperature for 1 h.7) Read FP on Envision.8) Calculation formula for original data:%Inhibitionrate=(FPV-FPS)/(FPV-FPB)×100% |
| Affinity data for this assay | |
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