| Assay Method Information | |
| | Quantification of the Phosphorylated Product Formed from the Kinase Reaction in Presence of ATP |
| Description: | Serially diluted compounds were incubated for 60 minutes at room temperature in the presence of ATP/P33 gammaATP mix, substrate and enzyme in a volume of 15 microL of kinase buffer in 384-well plates (Thermo Scientific, cat #4312).The reaction volume contains, in final concentration, 52 microM ATP, 8 nM PERK and 300 microM substrate in 50 mM Hepes pH 7.5, 3 mM MgCl2, 1 mM DTT, 3 microM Na3VO4, 0.2 mg/ml BSA. The final concentration of DMSO was 1%.At the end of the incubation, an amount of 60 microL of Dowex resin (Sigma, customized resin 1×8 200-400 mesh cat #13858-U) in 150 mM sodium formate buffer pH=3.0 was added to stop the reaction and capture unreacted ATP/P33 gammaATP, separating it from the phosphorylated substrate in solution. After 60 minutes of rest, a volume of 22 microL of supernatant was transferred into 384-Optiplates (Perkin-Elmer, cat #6007290). After the addition of 50 microL of Microscint 40 (Perkin-Elmer, cat #6013641), the radioactivity was counted in the TopCount (Perkin Elmer).The data per each molecule are analyzed by an internally customized version of the SW package Assay Explorer , or by GraphPad Prizm software alternatively, 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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