A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA

Adrian Velazquez-Campoy, Bárbara Claro, Olga Abian, Jonas Höring, Louis Bourlon, Rafael Claveria-Gimeno, Eric Ennifar, Patrick England, Jonathan Brad Chaires, Di Wu, Grzegorz Piszczek, Chad Brautigam, Shih Chia Tso, Huaying Zhao, Peter Schuck, Sandro Keller, Margarida Bastos

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called “stoichiometry” (n), a concentration-correction factor.

Original languageEnglish (US)
Pages (from-to)429-451
Number of pages23
JournalEuropean Biophysics Journal
Issue number3-4
StatePublished - May 2021


  • Benchmark study
  • Data treatment
  • Isothermal Titration Calorimetry (ITC)
  • Ligand-binding
  • Sample preparation
  • Standard reaction

ASJC Scopus subject areas

  • Biophysics


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