| Assay Method Information | |
| | IRE1alpha TR-FRET Competition Binding Assay |
| Description: | A His-tagged IRE1α 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 0.25 nM) was pre-incubated with anti-His Europium labeled antibody (Life Technologies PV5596, final concentration 2 nM) for one hour at 4° C. in 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. An Alexa fluor 647-labeled probe based on an ATP competitive inhibitor was added to a final concentration of 2 nM. Reactions were carried out for one hour at room temperature in a final volume of 20 μL in 384 well white ProxiPlates (Perkin Elmer 6008289). Binding of the probe to the IRE1α protein 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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