CHEMBL3705660

High Throughput Screening Assay: The purified Na/K-ATPase was prepared from pig kidney. The specific activities of Na/K-ATPase of various kidney preparations were in the range of 900-1,200 umol/mg/h, which were greater than 95% of the total ATPase activity. The high throughput screen is conducted in a 96-well format with the final reaction volume of 100 ul containing the following components: 100 mM NaCl, 20 mM KCl, 1 mM MgCl2, 1 mM EGTA, 20 mM Tris-HCl (pH 7.4) and 0.2 ug of purified Na/K-ATPase. After compounds were added, mixtures were incubated for 15 min at 37 C. for 15 min and reaction was initiated by adding 2 mM ATP.Mg mixture.Reactions were carried out for 15 min and then stopped by the addition of 100 ul ice-cold trichloroacetic acid. Reaction mixtures were cleared by centrifugation, and assayed for released phosphate using the BIOMOL GREEN Reagent according to the manufacturer's instructions. In addition, the control Na/K-ATPase activity was measured.