| Assay Method Information | |
| | Human PDE-5A1 Inhibition Assay |
| Description: | Enzymes and SubstratesCatalog Enzyme Enzyme Used Assay # Lot # (ng/reaction) SubstratePDE5A1 60050 181008-G 0.2 100 nM FAM-cGMPThe serial dilution of the compounds was first performed in 100% DMSO with the highest concentration at 1 mM and 0.1 mM. Each intermediate compound dilution (in 100% DMSO) will then get directly diluted 10× fold into assay buffer for 10% DMSO and 5 μL of the dilution was added to a 50 μL reaction so that the final concentration of DMSO is 1% in all reactions.The enzymatic reactions were conducted at room temperature for 60 minutes in a 50 μL mixture containing PDE assay buffer, 100 nM FAM-cGMP, a PDE enzyme (Table 2) and the test compounds.After enzymatic reaction, 100 μL of a binding solution (1:100 dilution of the binding agent with the binding agent diluent) was added to each reaction and the reaction was performed at room temperature for 60 minutes.Fluorescence intensity was measured at excitation of 485 nm and an emission of 528 nm using a Tecan Infinite M1000 microplate reader. |
| Affinity data for this assay | |
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