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Assay Method Information |
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| | Thermal Shift Assay (TSA) |
| Description: | In a typical TSA, 0.4 μg of BRD4 BD1 protein were mixed with 5× Sypro Orange (Molecular Probe) and adjusted to a final volume of 5 μl with 100 mM HEPES buffer, pH 7.5 (Applichem), and 150 mM NaCl (Sigma). For binding experiments, 1% DMSO (Sigma) or JQ1 in serial dilution (0.14 μM to 100 μM, 3-fold) was added to the mixture. To protect samples from evaporation, they were overlaid with 1 μl of silicone oil DC200 (Sigma). Melting curves were recorded using a ThermoFluor system (Johnson and Johnson Pharmaceutical Research and Development) after heating the samples from 25 up to 95 °C while measuring the fluorescence intensity of the dye with the excitation and emission filters set to 465 and 590 nm, respectively. |
| Affinity data for this assay | |
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