Jul 07, 2025
Isothermal Titration Calorimetry (ITC)
- Dieter Waschbuesch1,
- Amir Khan2
- 1School ofBiochemistry and Immunology, Trinity College Dublin;
- 2Trinity College Dublin
- AKhanLab
Protocol Citation: Dieter Waschbuesch, Amir Khan 2025. Isothermal Titration Calorimetry (ITC). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkr7x6v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 26, 2025
Last Modified: July 07, 2025
Protocol Integer ID: 221125
Keywords: isothermal titration calorimetry, peptide, itc, enthalpy, thermodynamic parameters such as enthalpy, quantitative data on affinity, protein, interacting protein, underlying thermodynamic parameter
Funders Acknowledgements:
Research Ireland
Grant ID: 20/FFP-A/8446
Abstract
This protocol describes in general our laboratory’s way how to perform ITC between two interacting proteins or a protein and a peptide. It is a useful method to get quantitative data on affinity and the underlying thermodynamic parameters such as enthalpy.
Materials
Consumables
- 1.5 ml reaction tubes
- 0.5 ml PCR tubes
- Dialysis tube 12 kDa MWCO (Sigma, D9777)
- Pur-A-Lyzer™ Midi Dialysis tubes 1 kDa MWCO (Sigma, PURD10005-1KT) for peptides
- Millex -GP Syringe filters (Merck, SLGP033RS)
Reagents
- Purified proteins/peptides (protein A, protein/peptide B)
- Dialysis Buffer (20 mM Tris pH 8.0 at 4ºC, 150 mM NaCl, 1 mM DTT)
Note
Some proteins require the presence of additional co-factors, e.g. Rab GTPases require the presence of Mg2+, which is added to the buffer in case an ITC experiment involves a Rab-GTPase. Different buffers also work, it is just important that the two interactors are dialysed into the same buffer.
Equipment
- Calorimeter ITC 2000 (MicroCal)
- 1 l laboratory beaker
- PC with the MicroCal ITC200 software (version 1.30, Malvern) and Origin 7 (OriginLab Corporation)
- Tabletop microcentrifuge (VWR MicroStar 12)
Troubleshooting
Protocol
Dialyse the proteins/peptide against dialysis buffer. For proteins, conventional dialysis tube is used, for small peptides the Pur-A-lyzer 1 kDa MWCO is used according to the manufacturer’s instructions. Stir using a magnetic stirrer at 100-200 rpm (gentle in other words). Dialysis time should be at least 2 hours, if the buffer mismatch is greater, longer times are recommended (e.g. overnight at 4°C).
Note
To avoid buffer mismatch, the two proteins need to be dialyzed against the dialysis buffer in the same beaker.
Determine noise of the ITC instrument by titrating water into water with standard 'Noise' injection parameters from MicroCal (temperature, spin, injection volumes, etc) . A noise level <1.5 microCal/sec is deemed acceptable and the experiment will likely succeed.
Determine the concentration of the proteins or peptide and adjust volumes to the desired concentration (e.g. 500 µl of 40 µM protein A for the cell and 200 µl of 400 µM protein/peptide B).
Note
Concentrate proteins higher than the desired concentration. Filter some dialysis buffer to dilute proteins to the desired concentration.
A good starting point is for titrations is 40 µM of protein A in the cell of the instrument and 400 µM protein/peptide B in the syringe to titrate into the cell. The exact individual parameters need to be determined in the course of conducting the experiments.
Directly before the experiment, centrifuge proteins/peptide for 3-5 minutes at 12,300 x g.
Note
It can be beneficial to de-gas the protein sample for the cell before that spin.
Carefully fill the syringe to titrate into the cell with protein/peptide B (400 µM). About 40 µl is required for syringe, but practically, 80 µl minimum is required to insure filling.
Load ~350 µl protein A (40 µM) with a Hamilton syringe and inject smoothly into the sample cell of ITC200 (200 µl) to insure complete fill. Avoid bubbles.
Place injector into sample cell and run the MicroCal ITC200 software. Check the instrument and run settings:
- Reference power: 5 µcal/sec
- Temperature: 20°C
- Stir speed: 750 rpm
- Initial delay: 60 sec
- injection spacing: 180 sec
- Injection parameters: 22* injections, 2.0* µl each, 4* second duration, spaced by 180 sec. Initial inject was 0.5 µL with a duration of 1 sec.
Note
Parameters marked with an * are adjusted individually for the different protein/peptide combinations.
Run protocol in the software.
Analyse the data in Origin with the manufacturers data analysis module.