| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | ITC experiments were conducted on a MicroCal Auto-ITC200 system (GE Healthcare life sciences). All titration experiments were performed at 20° C. in ITC buffer (10 mM Tris, 150 mM NaCl, pH 7.5). hHint1 was exchanged into ITC buffer using Micro biospin6 columns (BioRad, USA) and final protein concentrations were determined as described above. To determine the dissociation constant of stock concentration (300-400 uM) of inhibitors was titrated with 15-20 uM of Hint1. Twenty injections of ligand were injected (injection volume 2 ul) into the protein cell. The resulting change in enthalpy was measured and the background heat of dilution was subtracted by performing similar experiments in the absence of inhibitors. The background heat of dilution was subtracted from the resulting data and was fitted into one-site binding model using the ITC200 microcal software. The resulting association constant obtained by fitting the curve was converted into Kd using Ka=1/Kd relationship. |
| Affinity data for this assay | |
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