Isothermal titration calorimetry (ITC)
Isothermal titration calorimetry (ITC) is a powerful technique to determine the thermodynamic parameters governing molecular interactions. During ITC experiments a concentrated ligand is titrated into a solution containing its binding partner during a series of small-volume injections. Complex formation between molecules either results in the uptake or release of heat that can be directly measured by ITC. This allows for the determination of the dissociation constant (Kd), the stoichiometry of the interaction (n) and the enthalpy of binding (ΔH), and enables us to calculate the Gibbs free energy (ΔG) and entropy (ΔS). Typically, it is possible to analyse interactions with Kd values from the mM to the nM range.
Isothermal titration calorimetry is used to measure the strength of molecular interactions. Typically, it is possible to analyse interactions with Kd values from the low mM to the nM range.
Both molecules must be in identical buffer conditions. This is best achieved by dialysing both macromolecules exhaustively against an appropriate buffer (in the same beaker). For small molecule ligands the final dialysis buffer can be used to make up the ligand solution.
• Cell: 300 μl of 10-100 μM
• Syringe: 40 μl of 10-20x the molar cell concentration
• Microcal PEAQ-ITC (Malvern)
• Microcal iTC200 (GE, now Malvern Instruments)
UK Biological Microcalorimetry Facility (Prof. Alan Cooper, Univ. of Glasgow)