| Assay Method Information | |
| | Isothermal Titration Calorimetery |
| Description: | ITC was performed using a ITC200 microcalorimeter from GE (Northampton, Mass.). All experiments were carried out at 25° C. while stirring at 1000 rpm, in ITC buffer (50 mM HEPES pH 7.4 at 25° C., 150 mM NaCl). The microsyringe was loaded with a solution of the protein sample (225 μM, in ITC buffer). The compound solution (22.5 μM, in ITC buffer) was titrated into the protein solution via syringe. All titrations were conducted using an initial injection of 0.2 μl, followed by 19 identical injections of 2 μl with a duration of 5 sec (per injection) and a spacing of 90 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. The collected data were implicated in the MicroCal Origin software supplied with the instrument to yield enthalpies of binding (ΔH) and binding constants (Ka). The collected data were implicated in the MicroCal Origin software supplied with the instrument to yield enthalpies of binding (ΔH) and binding constants (KB) as previously described by Wiseman and coworkers. Thermodynamic parameters were calculated (ΔG=ΔH−TΔS=−RT ln KB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). A single binding site model was employed. |
| Affinity data for this assay | |
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