Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Prepare biological macromolecules and ligands with appropriate concentrations and buffer conditions. Ensure samples are properly degassed and free of particulates before loading into the ITC instrument.

Load the macromolecule into the sample cell and ligand into the syringe. Set experimental parameters including temperature, injection volume, and timing to monitor binding thermodynamics.

Process raw calorimetric data to determine binding affinity (Kd), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) values. Use integrated peak analysis and curve fitting to characterize macromolecule-ligand interactions.

Review typical ITC thermograms and binding isotherms from moderate-affinity biological systems. Interpret results to understand binding thermodynamics and validate experimental methodology.