Protein Crystallography and Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology (ISMB)
Birkbeck College / University College London

Isothermal Titration Calorimeter
(VP-ITC, Malvern)


Isothermal titration calorimetry measures the heat that is released (or required) when molecules interact. In a typical ITC experiment, a single titration of ligand into a solution of its interaction partner results in a series of peaks representing the heat of each addition. These can be analysed to give a complete characterization of binding thermodynamics, i.e. the binding affinity, stoichiometry, enthalpy and entropy.

A wide range of buffer conditions are suitable. A further advantage is that molecules do not need to be labelled or altered, so the method lends itself to investigations at native conditions. However, a relatively large sample volume is required, so alternatively Surface Plasmon Resonance could be used, or, if one of the binding partners is fluorescent, anisotropy measurements could be performed.

Sample Requirements
  • Volumes: at least 2.5 ml of sample, 2 times 600 μl of titrant/ligand.
  • Concentrations: depends on expected association constant and heat generated by the reaction, usually 10-200 μM for sample, 10-20x greater for ligand for 1:1 binding
  • Sample and ligand must be extensively dialysed into the same batch of buffer for the experiment. Please get in touch if this is impossible. ITC is very sensitive against buffer mismatches.
  • Stability at required concentration and temperature must be tested beforehand as precipitation will make results impossible to interpret

Buffer Requirements
  • pH ≥ 5 (instrument may be damaged by acid).
  • Avoid Tris buffer.
  • Avoid DTT,- avoid TCEP and bME, although ~1 mM may be acceptable.
  • Keep plenty of the dialysis buffer for diluting, cell priming and control run.

  • It is important to optimize the concentration in order to get fittable data.
  • Sample concentration: use estimated association constant KA and stoichiometry N and the following rule:
          5 ≤ KA·Csample·N ≤ 500, for biological samples KA·Csample·N = 100 is advisable,
          e.g. suppose N=1 and KA is 105 M-1, then C=5/(1·105 M-1)=5·10-5 M= 500 μM. In any case it should be ≥50 μM.
  • Expected data can be simulated in advance using ITC Expert software.
  • Ligand: if possible 15 times the concentration of sample

Temperature Dependence of ΔH
  • ΔH is temperature dependent, hence could be zero at chosen temperature, ideally ΔH should be 2-10 μCal/s for the first few injections.
  • Temperature and/or concentrations may have to be changed for better signal
  • Concentrations can be 3-5 times lower if signal-to-noise ratio is acceptable.

Please contact us for detailed protocols and planning your experiment.

ISMB Protein Crystallography and Biophysics Centre, Birkbeck, University of London
Last modified April 2021