| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | Compounds were 3-fold serially diluted in order to obtain from 3.333 to 0.000169 microM final concentration, then incubated for 60 minutes at room temperature in the presence of ATP, substrate and enzyme in a final volume of 15 microL of kinase buffer in 384-well plates (Perkin Elmer cat. #6007290).The final concentration of the different reagents is 52 microM ATP, 8 nM PERK, 300 microM substrate, 50 mM Hepes pH 7.5, 3 mM MgCl2, 1 mM DTT, 3 microM Na3VO4, 0.2 mg/ml BSA, 1% DMSO.After 60 minutes, an equal volume (15 microL) of ADP-Glo Reagent was added to each well to terminate the kinase reaction and deplete the remaining ATP. After 40 minutes, 30 microL of Kinase Detection Reagent is added, which simultaneously converts ADP to ATP and allows the newly synthesized ATP to be measured using a coupled luciferase/luciferin reaction. After further 40 minutes luminescence was read by ViewLux Instrument (Perkin Elmer). The data are analyzed by GraphPad Prism software which provides sigmoidal fittings of the curves for IC50 determination using a 4 parameter logistic equation:y=bottom+(top−bottom)/(1+10{circumflex over ( )}((log IC50 −x)*slope))where x is the logarithm of the inhibitor concentration, y is the response; y starts at bottom and goes to top with a sigmoid shape. |
| Affinity data for this assay | |
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