| Assay Method Information | |
| | In vitro RNase Assay |
| Description: | 5′-Carboxyfluorescein (FAM)- and 3′-Black Hole Quencher (BHQ)-labeled XBP1 single stemloop mini-substrate (5′FAM-CUGAGUCCGCAGCACUCAG-3′BHQ) were purchased fromDharmacon We incubated 0.25 μM IRE1α* or dP-IRE1α* with inhibitors or DMSO for 30 min in assay buffer (20mM Tris at pH7.5, 50mM sodium chloride, 1mM magnesium chloride, 2mM DTT, 0.05% Triton X-100 (v/v)), followed by incubation with 1 μM RNA substrate for 10 min. The reaction was quenched by adding urea to a final concentration of 4 M, and the fluorescence was detected on a SpectraMax M5 microplate reader (Molecular Devices) or a Perkin Elmer 2104 Envision microplate reader with excitation and emission wavelengths of 494 nm and 525 nm, respectively. The fluorescence intensities were normalized by setting the signal for the reaction without IRE1α* to 0. |
| Affinity data for this assay | |
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