| Assay Method Information | |
| | IRE1α TR-FRET Competition Binding Assay |
| Description: | To determine the affinity of compound binding to the kinase domain of IRE1 alpha, a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) competition assay was used. A His-tagged IRE1 alpha kinase dead construct containing the kinase and RNase domains (KR, AA G547-L977, D688N) was expressed in Sf9 insect cells. The purified protein (final concentration 0.006 μM micromolar) was pre-incubated with anti-His Europium labeled antibody (Life Technologies PV5596, final concentration 0.002 μM micromolar) for one hour at 4° C. in 1X TR-FRET Assay Buffer (50 mM HEPES, pH 7.5.10 mM MgCl2, 0.083 mM Brij 35, 1 mM DTT, and 0.1% bovine gamma globulin) prior to addition to test compounds. A fluorescent labeled probe based on an ATP competitive inhibitor (Kinase Tracer 236, Life Technologies PV5592) is added to a final concentration of 0.1 μM (micromolar). Reactions were carried out for one hour at room temperature in a final volume of 20 μL (microliter) in 384 well white ProxiPlates (Perkin Elmer 6008289). Binding of the tracer to the IRE1 protein alpha was detected in an Envision instrument (PerkinElmer) equipped with a TRF laser option and a LANCE/Delfia Dual/Bias D400/D630 mirror (Ex 347 nm, 1st Em 665 nm, 2nd Em 615 nm). |
| Affinity data for this assay | |
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