Faculty of Biological Sciences

Isothermal Titration Calorimetry (ITC)

ITC titrates one molecule into a solution of a "receptor" measuring the small heat change associated with binding (or ligand dilution). This gives information about equilibrium affinity, enthalpy and stoichiometry. This can be used to determine entropic contributions to interactions. This thermodynamic information complements the kinetic binding information from SPR.

ITC can be used to analyse protein-protein, protein-small molecule, protein-nucleic acid or essentially any interaction. No immobilisation or labelling is required.

Simple Description

ITC is used to analyse the interactions between molecules. It measures the small heat changes as molecules interact (or as solutions are diluted). Addition of substoichiometric aliquots of a ligand to a receptor in the ITC cell gives a "spike" of heat as the molecules interact – see the middle panel above. As the receptor becomes saturated, the size of each spike diminishes until only the heat of ligand dilution is seen. These data can be transformed to give a binding curve (right hand panel). Fitting these data to a binding model gives values for Kd, stoichiometry and enthalpy. Further analysis can then give about the entropic contribution to binding. The thermodynamic description of an interaction can be important in screening a range of ligands for receptor binding.

The advantages of ITC are that it is label-free (requiring no modification of either binding partner) and that reactions are performed in solution, i.e. without immobilisation on a surface (c.f. SPR).

Use this when…

ITC is used for binding studies in solution (protein-protein, protein-nucleic acid or small molecule interactions). It can also be used for enzyme kinetic analysis.

Related techniques

AUC and SPR can be used to monitor interactions between molecules. SPR is able to determine kinetic rate constants (but not thermodynamics parameters) and requires immobilisation of one partner which can be problematic.

Practical Considerations

ITC requires about 1.8 ml of typically 10-100μM solution to fill the sample cell. The injection syringe holds 250μl of the titrant, at a concentration e.g. 7-fold higher than the molecule in the cell. Choose concentrations of molecules based on the strength of the interaction and the heat change, to give a signal larger than that from ligand dilution. It is important to use pure reagents at known concentrations to get the best results.