Assay Method Information | |
| Inhibitory Activity Against KHK In Vitro |
Description: | Experiment of Inhibitory Activity Against KHK In Vitro: With the involvement of ATP, fructokinase phosphorylates fructose to produce fructose-1-phosphate and ADP. In this experiment, the effect of the compound on the activity of fructokinase was determined by measuring the amount of ADP produced in the process. Test compound stocks were prepared in DMSO. Before use, the stocks were 3-fold serially diluted with assay buffers (25 mM HEPES, 300 mM KCl, 10 mM MgCl2, 10 mM CaCl2), pH 7.0) to 5-fold the final concentration tested, and the DMSO content was adjusted to 1%. In a 384-well plate, the test compound was added to achieve a final concentration of 7.6 nM to 100 μM (assay buffers containing 1% DMSO were added to background and control wells). 5 ng/μL purified hKHK-C (assay buffers were added to background wells), 50 mM fructose and 0.2 mM ATP were added to reach a total volume of 10 μL and reacted at room temperature for 30 min. After the reaction was completed, 10 μL of ADP-glo (promega) was added and mixed uniformly, and the plate was incubated at room temperature for 40 min. Then the ADP-glo was added again, mixed uniformly and reacted for 40 min. The spontaneous luminescence intensity was measured using an Envision microplate reader. |
Affinity data for this assay | |
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