| Assay Method Information | |
| | GAK pSENs Assay |
| Description: | This example describes one illustrative method for determining the IC50 of the compounds of the present invention against GAK (amino acids Q25-N335). GAK protein was dissolved at 160 nM in 1× enzyme solution (50 mM HEPES pH7.5, 10 mM MgCl2, 1 mM DTT, 1% glycerol, 0.5 mM EGTA, 0.01% Briji35, 0.02% BSA) and 12.5 ul was incubated with 250 nl DMSO or 250 nl compound in DMSO at 25° C. for 15 min. The reaction was started by addition of 12.5 ul of 30 uM AQT0766 (Assayquant, CSKS-AQT0766B) and 140 uM ATP which were diluted in 1× substrate dilution buffer (50 mM HEPES pH7.5, 10 mM MgCl2, 1 mM DTT, 1% glycerol, 0.5 mM EGTA, 0.01% Briji35). The rate of product formation (fluorescence signal with Excitation at 360 nm and Emission at 486 nm, Molecular Devices SpectraMax Paradigm) was measured every 2 minutes for 120 minutes at 25° C., and the RFU data was analyzed by linear regression. For IC50 determination, rates normalized relative to uninhibited controls were plotted against compound concentration and fitted using a 4 parameter non-linear regression curve fit (Y=Bottom+((Top−Bottom)/(1+((IC50/X){circumflex over ( )}Slope))), XLfit Model 205). |
| Affinity data for this assay | |
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