Jun 09, 2025
Isothermal Titration Calorimetry (ITC)
This protocol is a draft, published without a DOI.
- Dieter Waschbüsch1,
- Amir R. Khan1
- 1School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland
Protocol Citation: Dieter Waschbüsch, Amir R. Khan 2025. Isothermal Titration Calorimetry (ITC). protocols.io https://protocols.io/view/isothermal-titration-calorimetry-itc-g2u8byezx
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 08, 2025
Last Modified: June 09, 2025
Protocol Integer ID: 219776
Keywords: ASAPCRN
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 thermodynamics.
Guidelines
General considerations
- To avoid buffer mismatch, the two proteins need to be dialysed against the same buffer in the same beaker.
- Concentrate proteins above the desired concentration. Filter some dialysis buffer to dilute proteins to the desired concentration.
- A good starting point 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.
Materials
1. Materials
1.1. Consumables
- 1.5 ml reaction tubes
- 0.5 ml PCR tubes
- Dialysis tube 12 kDa MWCO (Sigma, D9777) or Pur-A-Lyzer™ Midi Dialysis tubes 1 kDa MWCO (Sigma, PURD10005-1KT) for peptides
1.2. 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)
1.3. 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)
Procedure
Procedure
5m
5m
Pre-wet dialysis tube with distilled water in a small beaker.
Close dialysis tube on one end with clamp.
Fill in the protein A solution.
Close the dialysis tube with another clamp.
Repeat for the protein/peptide B or use a Pur-A-Lyzer tube (peptide) according to the manufacturer’s instructions.
Put the filled dialysis tubes in a swimmer in the beaker and dialyze protein A and protein/peptide B against the buffer. Stir using a magnetic stirrer at 100-200 rpm (gentle in other words). Dialysis should be at least for 2 hours, if the buffer mismatch is greater, longer times are recommended (e.g. overnight at 4°C).
Determine noise of the ITC instrument by titrating water into water.
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).
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.
5m
Carefully fill the syringe to titrate into the cell with protein/peptide B (400 µM). About 100 µl is required.
Add ~300 µl protein A (40 µM) with a Hamilton syringe into the measuring cell. Avoid bubbles.
Check the instrument and run settings:
Reference power: 5 µcal/sec
Temperature: 20°C
Stir speed: 750 rpm
Initial delay: 60 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.
Analyse the data in Origin with the manufacturer module.