| Assay Method Information | |
| | In Vitro Kinase Activity Assay |
| Description: | Inhibitors (initial concentration of 10 or 60 μM, three-fold serial dilutions) were incubated with IRE1α* in cleavage buffer (20 mM HEPES, 0.05% Triton X-100 (v/v), 50 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 7.5) for 30 min, followed by incubation with 10 μCi [γ32P] ATP(3,000 Ci mmol−1, PerkinElmer) at 23 C. for 3 hours. Samples were then spotted onto glass fiber paper (Easytab-C Glass Fiber Filter Paper, Perkin Elmer) and washed in twice with 0.5% phosphoric acid and autoradiographed using a GE Typhoon FLA 9000 Imager. Blots were quantitated using ImageQuant software. The percent inhibition was quantified by setting the background (no kinase well) as 0 and standardizing to IRE1α* without compound treatment (IRE1α*+DMSO). Dose-response curves were fit using one-site fit logIC50 parameter using GraphPad Prism analysis software. |
| Affinity data for this assay | |
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