| Assay Method Information | |
| | KHK-C ADP-Glo Kinase Inhibitory Activity Assay |
| Description: | I. Experimental Procedures:1) The compounds were transferred to a 384 reaction plates (PE, 6007290) with Echo (Labcyte, 550);2) After centrifugation, a KHK—C buffer containing 1 nM KHK—C(Origene, TP323488) was added, and incubation was conducted for 15 min at 25° C.;3) A substrate mixture containing 200 μM D-Fructose (Sigma, F2543) and 100 μM ATP was then added, and incubation was conducted for 60 min at 25° C.;4) 10 μL of ADP-Glo (Promega, V9102) was added, incubation was continued for 60 mn, then 20 μL of Detection solution was added;5) After the incubation was continued for 60 min, the luminescence value was read with the Envision multifunctional microplate reader; and6) Finally, the IC50 (median inhibitory concentration) of each compound was obtained using the nonlinear fitting formula by means of the XLFIT software.Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogIC50−X)×HillSlope))X: Log value of compound concentrationY: Inhibition ratio (%)Inhibitionratio(%)=(Meanvalueofnegativecontrol-Readingofcompound)(Meanvalueofnegativecontrol-Meanvalueofpositivecontrol)*100%Negative control: DMSO |
| Affinity data for this assay | |
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