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Assay Method Information |
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| | Isothermal Titration Calorimetry (ITC) |
| Description: | ITC binding isotherms were recorded on a MicroCal ITC200 instrument (MalvernInstruments). Titrations were conducted at 25 °C by injecting aliquots (2.0 μL) of ligand solutions (1 mM) into protein samples (60.9 μM), both of which had been diluted in 50 mM Tris-HCl buffer (pH 7.5), containing 100 mM KCl, and adjusted to give a final concentration of 10% v/v DMSO-d6. Titrations were typically 20 injections at 90 s intervals. Small background heats from dilution of the ligand were subtracted after performing a second control titration of ligand samples into buffer without protein. Binding isotherms were integrated to give the enthalpy change of each injection and plotted against the molar ratio of ligand added to the sample cell. |
| Affinity data for this assay | |
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