Enables direct measurement of the thermodynamics of an association reaction. Most commonly used to measure the affinity, enthalpy (heat change) and stoichiometry of molecular interactions. In some cases, binding kinetics can also be determined. Typically, two component reactions are measured with one molecule titrated against the other. Any pair of molecules which interact or which are suspected of interacting can be used, e.g., protein:small molecule ligand, protein:protein, protein:DNA, DNA:DNA. The technique does not require any labelling or modification of materials and is largely independent of molecular size. Concentration dependent oligomerization of a single species may also be measured.
What are the sample requirements?
- Pure samples of known concentration are required.
- Concentrations are typically 10-20 μM protein into which 100-200 μM ligand is titrated. However, optimal concentrations are dependent on the affinity of the interaction and may be 10x lower or higher.
- Volumes are instrument dependent. A single ITC experiment and control will typically require 2.5 ml of protein solution and 1 ml of ligand solution on a VP-ITC instrument (Microcal/MalvernPanalytical), whereas an ITC200 or PEAQ instrument from the same manufacturer requires six-fold lower volume.
What other specific considerations are relevant?
- It is important that both components (e.g., protein and ligand) are perfectly matched in buffer composition to minimise heats of dilution (e.g., have been dialysed or undergone gel filtration in the same batch of buffer – which should be retained for dilution and control experiments).
- High concentrations of solubilizing agents (e.g., glycerol, DMSO) should be avoided.
- Reducing agents cause artefacts; DTT must be avoided and TCEP or β-mercaptoethanol should be < 5mM and preferably much lower.
- pH should be > 5 (instrument is degraded by acid).
- Both components should be stable for > 6 hours at room temperature.
Partners offering this technique
MOSBRI reference partner sites for this technique: