| Assay Method Information | |
| | PERK enzyme assays |
| Description: | The biochemical activity of compounds was determined by incubation with PERK recombinant enzyme (cytoplasmic domain corresponding to residues 540-1115) and elF2alpha peptide substrate (Aminoacid sequence: LLSELSRRRIRSINK SEQ ID Nr: 1; purchased from American Peptide Company, product #341923). This was followed by:Method 1: Quantification of the ADP Formed from the Kinase Reaction by ADP-Glo Kinase Assay (Promega Cat. 9102). Utra Pure ATP was Included into ADP-Glo Kinase Assay Kit.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 IC 50 −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.Method 2: Quantification of the Phosphorylated Product Formed from the Kinase Reaction in Presence of ATP (Aldrich, Cat #A2620-9).Serially diluted compounds were incubated for 60 minutes at room temperature in the presence of ATP/P33gammaATP 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/P33gammaATP, 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). |
| Affinity data for this assay | |
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