| Assay Method Information | |
| | STAT6 Assay |
| Description: | HTRF binding assays were perfonned using 0.15 nM biotinylated truncated STAT6 (123-632)-avi purified from E.coli, IX Streptavidin-terbium (CisBio) prepared by mixing SA-Tb in PPI detection buffer (CisBio), 20 nM proprietary fluorescein-labeled probe, and test compounds in assay buffer consisting of 50 rnM HEPES-Na pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.1% Tween-20 with a final volume of 20 uL. Compound stocks were dissolved at 10 mM in 100% DMSO and 11 point titration with 3 fold serial dilution was perfonned in white, opaque 384 well microplates. Reaction plates were incubated at room temperature for 30 minutes. Plates were centrifuged at low rpm for 5 mins, and the ratio of fluorescence intensities were measured at emission wavelengths for fluorescein acceptor (520 nm) and terbium donor (495 nm) on Envision Plate reader. % Inhibition was calculated from the 520/495 ratio generated by using proprietary positive control compound for 100% inhibition and DMSO only reactions for 0% inhibition. Data was processed and dose response curves were generated using GraphPad Prism to determine the concentration required for inhibiting 50% of the HTRF signal (IC50). |
| Affinity data for this assay | |
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