| Assay Method Information | |
| | Kinase Assay |
| Description: | Inhibitors (initial concentration 10 or 60 μM, three-fold serial dilutions) were incubated with IRE1α* in cleavage buffer (20 mM HEPES at pH 7.5, 0.05% Triton X-100 (v/v), 50 mM potassium chloride, 1 mM magnesium chloride, 1 mM DTT) for 30 min, followed by incubation with 10 μCi [γ-32P] ATP(3,000 Ci mmol−1, PerkinElmer) at 23 °C for 3 hours. Samples were then spotted onto phosphocellulose paper and washed in triplicate with 0.5% phosphoric acid and autoradiographed. The percent inhibition was quantified by setting the background as 0 and standardizing to IRE1α* without compound treatment. |
| Affinity data for this assay | |
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