Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Biochemistry 4 Biopolymers University of Bayreuth Universitaetsstr 30 95447 Bayreuth Germany

Biomolecular Analysis Core Facility University of Manchester Oxford Rd Manchester M13 9PL UK

BioMolecular Screening and Protein Technologies Unit Centre for Genomic Regulation Dr Aiguader St 88 08003 Barcelona Spain

Biophysics Lab Protein Expression and Purification Core Facility EMBL Heidelberg Meyerhofstraße 1 69117 Heidelberg Germany

Biophysics Platform Novo Nordisk Foundation Center for Protein Research University of Copenhagen 2200 Copenhagen Denmark

Biozentrum University of Basel Klingelbergstrasse 50 70 4056 Basel Switzerland

Center for Molecular Biophysics UPR 4301 CNRS Orléans Rue Charles Sadron 45071 Orléans France

Departamento de Bioquímica y Biología Molecular y Celular Institute of Biocomputation and Physics of Complex Systems Universidad de Zaragoza C Mariano Esquillor S N 50018 Zaragoza Spain

Department of Biochemistry Faculty of Science Charles University Hlavova 8 128 43 Prague Czech Republic

Department of Biochemistry Netherlands Cancer Institute Plesmanlaan 121 1066CX Amsterdam Netherlands

Department of Biochemistry University of Bayreuth Universitätsstr 30 95447 Bayreuth Germany

Department of Biochemistry University of Oxford South Parks Rd Oxford OX13 5LA UK

Department of Biological Sciences BiophysX Centre Institute of Structural and Molecular Biology Birkbeck University of London Malet Street London WC1E 7HX UK

Department of Biological Sciences ISMB BiophysX Centre Institute of Structural and Molecular Biology Birkbeck University of London London WC1E 7HX UK

Department of Biology Bioscience Technology Facility University of York Wentworth Way York YO10 5DD UK

Department of Biotechnology and Biomedicine Technical University of Denmark Søltofts Plads Kgs 2800 Lyngby Denmark

Department of Biothermodynamics and Drug Design Life Sciences Center Institute of Biotechnology Vilnius University Sauletekio StSaulėtekio 7 10257 Vilnius Lithuania

Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry Hajdrihova 19 1000 Ljubljana Slovenia

Departments of Biophysics and Microbiology UT Southwestern Medical Center Dallas TX 75390 USA

Division of Biochemistry Department of Chemistry Faculty of Science University of Zagreb Horvatovac 102a 10000 Zagreb Croatia

EMBL Hamburg Notkestrasse 85 22607 Hamburg Germany

Faculty of Chemistry Chair of Drug and Cosmetics Biotechnology Warsaw University of Technology ul Noakowskiego 3 00 664 Warsaw Poland

Glycobiochemistry and Biomolecular Interaction and Crystallization Core Facility CEITEC MU Kamenice 5 625 00 Brno Czech Republic

Institut de Biologie et Chimie des protéines CNRS Université de Lyon 7 passage du Vercors 69367 cedex 07 Lyon France

Institut de Chimie Organique et Analytique CNRS FR 2708 UMR 7311 Université d'Orléans Orléans France

Institut de Recherches Servier 125 Chemin de Ronde 78290 Croissy sur Seine France

Institute of Biochemistry and Biophysics PAS Pawinskiego 5a 02 106 Warsaw Poland

Institute of Biocomputation and Physics of Complex Systems Universidad de Zaragoza C Mariano Esquillor S N 50018 Zaragoza Spain

Institute of Molecular Cell and Systems Biology University of Glasgow B4 13 Joseph Black Building G12 8QQ Glasgow Scotland UK

Integrated Structural Biology Grenoble 71 avenue des Martyrs 38042 Grenoble Cedex 9 France

Max Planck Institute of Biochemistry Am Klopferspitz 18 Martinsried 82152 Planegg Germany

Molecular Biophysics Institut Pasteur 25 28 Rue du Dr Roux 75015 Paris France

Molecular Biophysics Research Laboratory Departamento de Química UCIBIO Requimte Faculdade de Ciências e Tecnologia UNL Campus Caparica 2829 516 Costa da Caparica Portugal

MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK

NHLBI Biophysics Core Facility NHLBI NIH 50 South Dr Bethesda MD 20892 USA

ProteinTechnology Vienna Biocenter Core Facilities GmbH Dr Bohr Gasse 3 1030 Vienna Austria

Shared Research Facilities The Institute of Cancer Research London SW7 3RP UK

Eur Biophys J. 2021 Dec;50(8):1159-1160. doi: 10.1007/s00249-021-01573-x. PubMed

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