| Assay Method Information | |
| | Inhibition of Kinase Activity of IRE1alpha |
| Description: | The kinase reactions were performed in 384 well white ProxiPlate-384 Plus plates (PERKIN Elmer 6008280) using 25 mM MOPS assay buffer with 1 mM dithiothreitol, 25 mM MgCl2, 12.5 mM β-glycerophosphate, 5 mM EGTA, and 50 g/mL BSA. Test compounds were prepared on the day of assay and dispensed using D300 digital dispenser as a 10-point log dilution series in duplicate, normalized to a final DMSO concentration of 3%. Test compounds were pre-incubated for 30 min at room temperature with 10 nM IRE1α kinase (E31-11G from Signal Chem) in 2.5 L of assay buffer and the reaction started by addition of 2.5 L of ATP in assay buffer, to give a final ATP concentration of 100 m and 5 nM IRE1α kinase. After 4 hours incubation at room temperature the reactions were stopped and the kinase activity determined using the ADP-Glo™ reagent from Promega, according to the manufacturer’s instructions. Luminescence was measured on a luminometer (EnVision, PerkinElmer) and IC50 values calculated by fitting a sigmoidal curve to percent inhibition of control versus Log10 of compound concentration. |
| Affinity data for this assay | |
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