CHEMBL3706232

Caliper Mobility-Shift Assay: A sample to be tested is precisely weighed, dissolved by adding DMSO, mixed sufficiently to form 10 mM solution. The above mother solution is diluted with DMSO to 0.5 mM, then diluted in 3.162 gradient multiple to obtain total 11 concentrations.20 ul of substrate 10 uM FL-cGMP was added to 96-well plate, to which was added 1 ul of compound DMSO solution or compound-free DMSO solution, then was added 29 ul of 1.38 ng/ul PDE-5A enzyme buffer solution (100 mM Hepes pH 7.5, 5 mM MgCl2, 0.002% Brij-35). The maximum final concentration of the compound was 10 uM. After incubation at 30 C. for 1 h, 20 ul of 70 uM EDTA was added to terminate reaction. Substrate and products were separated and analyzed by electrophoresis.