| Assay Method Information | |
| | PERK In Vitro Activity Assay |
| Description: | In vitro Inhibition of PERK Enzyme Activity (isolated) Recombinant human EIF2AK2 (PKR) catalytic domain (amino acids 252-551), EIF2AK3 (PERK) catalytic domain (amino acids 536-1116), GFP-eIF2a substrate, and Terbium-labelled phospho-eIF2a antibody is obtained (Invitrogen, Carlsbad, CA).Express and purify HIS-SUMO-GCN2 catalytic domain (amino acids 584-1019) from E. coli. Perform TR-FRET kinase assays in the absence or presence of inhibitors in a reaction buffer consisting of 50 mM HEPES, pH 7.5, 10 mM MgCb, 1.0 mM EGTA, and 0.01% Brij-35, and 100-200 nM GFP-eIF2a substrate. PKR assays contain 14 ng/mL enzyme and 2.5 μM ATP (Km, −2.5 μM), PERK assays contain 62.5 ng/mL enzyme and 1.5 μM ATP (Km. app −1.5 uM), and GCN2 assays contain 3 nM enzyme and 90 μM ATP (Km, −200 uM). Add test compound, initiate the reaction by addition of enzyme, and incubate at room temperature for 45 minutes. Stop the reaction by addition of EDTA to a final concentration of 10 mM, add Terbium-labelled phospho-eIF2a antibody at a final concentration of 2 nM, and incubate for 90 minutes. Monitor the resulting fluorescence in an EnVison® Multilabel reader (PerkinElmer, Waltham, MA). Determine TR-FRET ratios and the resulting IC50 values using a 4-parameter nonlinear logistic equation as shown: Y=(A+((B−A)/(1+(C/x)AD)))) where, Y=% specific inhibition, A=Bottom of the curve, B=Top of the curve, C=absolute IC50 (concentration causing 50% inhibition), and D=hill slope. |
| Affinity data for this assay | |
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