| Assay Method Information | |
| | Isothermal Titration Calorimetry Analysis of Binding to Brd2-BD1 and Brd2-BD2 |
| Description: | Binding of compounds to BRD2 was monitored using an ITC200 (Microcal, Piscataway, N.J.). Either Brd2.1 (74-194) or Brd2.2 (348-455) was dialyzed overnight into a solution containing 50 mM HEPES, pH 7.0 and 150 mM NaCl at 15° C. Enzyme concentrations were determined after dialysis by absorbance at 280 nm using a molar extinction coefficient (ε280) of 25,565 M−1 cm−1 (2.1) or 16,055 M−1 cm−1 (2.2). Enzyme was diluted in dialysis buffer and 39.6 μL was loaded into the syringe. Compounds were dissolved in the same dialysis buffer and loaded in the cell to a volume of 204 μL. Injections were carried out by serial injections of enzyme; first, 1 injection of 1 μL, followed by 19 incremental injections of 2 μL, at 120 second intervals. Data from the first injection was excluded, due to pre-equilibration mixing between the contents of cell and syringe at the syringe tip. Peak areas were integrated, normalized, and then fitted by non-linear regression using the independent sites model in Origin (version 2.3.6, Microcal, Piscataway, N.J.). |
| Affinity data for this assay | |
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