| Assay Method Information | |
| | KHK Biochemical Assay |
| Description: | Compounds were tested for KHK enzyme inhibition in a high-throughput 384-well assay format using the ADP-Glo assay (Promega) in buffer consisting of 50 mM Hepes (pH 7.4), 140 mM KCl, 5 mM MgCl2 and 0.01% Triton-X. 0.2 nM. KHK-C or KHK-A enzyme was used in this assay with 0.5 mM (2×Km) ATP and 1.5 mM or 10 mM fructose (5×Km) for KHK-C and KHK-A, respectively. Compounds were serially diluted (1:3) in DMSO. The LabCyte ECHO Acoustic dispenser system was used to pre-spot the assay plates (384-well Non-Binding Surface plates, Corning, Catalog #3824) with 50 nL (200-fold final dilution) of compound.The compounds were pre-incubated with 5 μL of 2× final enzyme concentration for 30 minutes before adding 5 μL of 2× final concentration of ATP and fructose. The plates were incubated at room temperature for 4 hours before adding ADP-Glo reagent to quench the enzyme reaction, followed by incubation at room temperature for 1 hour. The ADP-Glo detection reagent was then added to the plates and luminescence was measured on the Envision plate reader after 1 hour. The addition of enzyme, substrates and ADP-Glo reagents to the plates was performed using the BioTEK EL406 liquid dispenser using the 5 μL dispensing cassette (BioTek 7170011). IC50 values were defined as the compound concentration that causes a 50% decrease in luminescence signal and were calculated using a sigmoidal dose-response model to generate curve fits. |
| Affinity data for this assay | |
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