| Assay Method Information | |
| | Inhibition of RNase Activity of IRE1alpha |
| Description: | The RNase reactions were performed in 384 well black ProxiPlate-384 Plus plates (PERKIN Elmer) using 50 mM Tris assay buffer with 0.5 mM MgCl2, 10 mM KCl, 0.03 % Tween, 2 mM DTT and 1% DMSO. 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 4%. Test compounds were pre-incubated for 30 min at room temperature with IRE1α kinase (E31-11G from Signal Chem) in 2.5 L of assay buffer. Then 2.5 l of assay buffer containing substrate (5′ Alexa Fluor 647 - rCrArUrGrUrC rCrGrCrArGrCrGrCrArUrG - Iowa Black RQ quencher 3′) (SEQ ID NO:1) added, giving a final concentration of enzyme of 0.325 nM and of substrate of 100 nM. After 20 minutes incubation at room temperature the reactions were stopped by added 5 L of 5 M urea, incubated at room temperature for 10 minutes and fluorescence measured on a plate reader (EnVision, PerkinElmer). IC50 values calculated by fitting a sigmoidal curve to percent inhibition of control versus compound concentration. |
| Affinity data for this assay | |
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