Assay Method Information | |
| ITC experiments (Isothermal titration calorimetry) |
Description: | ITC experiments were carried out on anAuto-ITC200 titration microcalorimeter (Malvern Instruments). All experimentswere carried out at 25 °C while stirring at 1,000 r.p.m. in ITC buffer(50 mM HEPES, pH 7.4, at 25 °C, 150 mM NaCl). The microsyringe wasloaded with a solution of the ligand sample. All titrations were conductedusing an initial injection of 0.4 μl followed by 19 identical injections of2.0 μl with a duration of 4 s (per injection) and a spacing of 120 s betweeninjections. The heat of dilution was determined by independent titrations(ligand into buffer) and was subtracted from the experimental data. Thecollected data were analyzed in the MicroCal Origin software supplied withthe instrument to yield enthalpies of binding (ΔH) and binding constants(KB) as previously described46. Thermodynamic parameters were calculated(ΔG = ΔH − TΔS = −RTlnKB, where ΔG, ΔH and ΔS are the changes in freeenergy, enthalpy of binding and ent |
Affinity data for this assay | |
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