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Macromolecular interactions in vitro, comparing classical and novel approaches

A Correction to this article was published on 27 April 2021

This article has been updated


Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( Twenty European students benefited from a week’s training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein–protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

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We thank members of Philippe Minard’s and Jean-Baptiste Charbonnier’s teams at I2BC for the sample preparation, Bruno Baron and Bertrand Raynal from Institut Pasteur, Paris for all their expert advices in molecular scale biophysics, and Eric Ennifar from Institut de Biologie Moléculaire et Cellulaire, Strasbourg for sharing its expertise in the study of biomolecular machineries using biophysical approaches. Friederike Möller and Hanna Müller-Landau from Dynamic Biosensors, Aymeric Audfray from Malvern Panalytical, Mathilde Belnou from NanoTemper technologies, and Stephanie Bourgeois and coworkers from Fluidic Analytics for their availability and all the fruitful discussion. We kindly thank all the participants to the MoSBio Training School, all the sponsors without whom this successful event had not been possible, and finally the keynote speakers, Julie Ménétrey and Terence Strick who shared their projects with us. Most of preparatory experiments were performed in the I2BC, PIM platform (, while some others were performed in Institut Pasteur, PFBMI platform. Finally, we thank ARBRE-MOBIEU network for its support to publish this article.


JBC is supported by ARC program (SLS220120605310), French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05, ANR-18-CE44-0008, ANR-20-CE11-0026, INCA 2016-1-PL BIO-11 and INCA SLX4 INCA 2016-159.

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PFV, PE, SU, CE, AVC, AR, JBC authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by CV, MAN, SU, PE, AVC, DS, GB, PS, CQ, CE, AR. The first draft of the manuscript was written by PFV and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Paloma Fernández Varela.

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The authors declare no competing interest. Pierre Soule (NanoTemper) and Christophe Quétard (FortéBio) helped during the training without commercial interest.

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The original online version of this article was revised: Due to corrections in Figures. In Fig. 2, the size of the subtitles a to g is reduced; In Fig 3b, “1DNA_1Ku70/Ku80” has been changed to “1DNA-1Ku70/Ku80” and In Fig 4, “50µg” has been changed to “50ng” in the MST cell at the bottom of the table.

Special Issue: COST Action CA15126, MOBIEU: Between atom and cell.

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Velours, C., Aumont-Nicaise, M., Uebel, S. et al. Macromolecular interactions in vitro, comparing classical and novel approaches. Eur Biophys J 50, 313–330 (2021).

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  • Molecular scale biophysics
  • Macromolecular interactions
  • Artificial binders
  • Double-stranded DNA breaks repair factors