Abstract
NanoTemper Monolith instruments have gained enormous popularity for measuring molecular interactions both in academia and industry. The underlying technology has been extensively reviewed along with its assumptions, limitations, and applications (Scheuermann et al., Anal Biochem 496:79–93, 2016). Several assumptions about the technique such as the extent of thermal deviations generated by the infrared laser and the thermophoretic foundation of the measured signal have been revised during the last decade. We present here in this letter the experience gathered in scientific service facilities about this technique and make scientists aware of possible pitfalls with the intention to promote knowledge and good practice throughout the scientific community.
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References
Alexander CG, Wanner R, Johnson CM, Breitsprecher D, Winter G, Duhr S, Baaske P, Ferguson N (2014) Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings. Biochim et Biophys Acta BBA Proteins Proteomics 1844(12):2241–2250. https://doi.org/10.1016/j.bbapap.2014.09.016
Baaske P, Wienken CJ, Reineck P, Duhr S, Braun D (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed 49(12):2238–2241. https://doi.org/10.1002/anie.200903998
Demchenko AP (2020) Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection. Methods Appl Fluoresc 8:022001. https://doi.org/10.1088/2050-6120/ab7365
Dhont JKG, Wiegand S, Duhr S, Braun D (2007) Thermodiffusion of charged colloids: single-particle diffusion. Langmuir 23(4):1674–1683. https://doi.org/10.1021/la062184m
Duhr S, Braun D (2006) Why molecules move along a temperature gradient. Proc Natl Acad Sci 103(52):19678–19682. https://doi.org/10.1073/pnas.0603873103
Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AEM, Ploegh HL (2013) Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc 8:1787–1799. https://doi.org/10.1038/nprot.2013.101
Hein CD, Liu XM, Wang D (2008) Click chemistry, a powerful tool for pharmaceutical sciences. Pharm Res 25:2216–2230. https://doi.org/10.1007/s11095-008-9616-1
Holmes MH (1995) Introduction to perturbation methods. Springer, New York
Horisawa K (2014) Specific and quantitative labeling of biomolecules using click chemistry. Front Physiol 5:457. https://doi.org/10.3389/fphys.2014.00457
Jarmoskaite I, AlSadhan I, Vaidyanathan PP, Herschlag D (2020) How to measure and evaluate binding affinities. Elife 9:e57264. https://doi.org/10.7554/eLife.57264
Jerabek-Willemsen M, André T, Wanner R, Roth HM, Duhr S, Baaske P, Breitsprecher D (2014) MicroScale thermophoresis: interaction analysis and beyond. J Mol Struct 1077(5):101–113. https://doi.org/10.1016/j.molstruc.2014.03.009
López-Méndez B, Baron B, Brautigam CA, Jowitt TA, Knauer SH, Uebel S, Williams MA, Sedivy A (2021) Reproducibility and accuracy of Microscale Thermophoresis in the NanoTemper Monolith—a multi laboratory benchmark study. Eur Biophys J
NanoTemper Technologies GmbH (2018) NanoPedia 2018—a part of the MO.Control 1.6 software package
NanoTemper Technologies GmbH (2020) NanoPedia 2020. https://nanotempertech.com/dianthus-nanopedia/. Accessed 23 Sept 2020
Ross D, Gaitan M, Locascio LE (2001) Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye. Anal Chem 73(17):4117–4123. https://doi.org/10.1021/ac010370l
Scheuermann TH, Padrick SB, Gardner KH, Brautigam CA (2016) On the acquisition and analysis of microscale thermophoresis data. Anal Biochem 496:79–93. https://doi.org/10.1016/j.ab.2015.12.013
Sedivy A (2021) Standard Operating Procedure for NanoTemper Monolith measurements. Eur Biophys J
Seidel SAI et al (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59(3):301–315. https://doi.org/10.1016/j.ymeth.2012.12.005
Sperling RA et al (2007) Size determination of (Bio)conjugated water-soluble colloidal nanoparticles: a comparison of different techniques. J Phys Chem 111:11552–11559. https://doi.org/10.1021/jp070999d
Theile CS, Witte MD, Blom AEM, Kundrat L, Ploegh HL, Guimaraes CP (2013) Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat Protoc 8:1800–1807. https://doi.org/10.1038/nprot.2013.102
Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100. https://doi.org/10.1038/ncomms1093
Wolff M, Mittag JJ, Herling TW, De Genst E, Dobson CM, Knowles TPJ, Braun D, Buell AK (2016) Quantitative thermophoretic study of disease-related protein aggregates. Sci Rep 6:22829. https://doi.org/10.1038/srep22829
Acknowledgements
Blanca López-Méndez acknowledges Professor Guillermo Montoya for his support. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Li Peng Lundgren acknowledges COST Action (CA15126) for a STMS (STMS 37745) at the Max Planck Institute of Biochemistry. As part of the Federal FTI strategy, this work was generously supported by the City of Vienna and the Austrian Ministry for Education, Science and Research. Arthur Sedivy acknowledges Peggy Stolt-Bergner for her support within VBCF ProTech. All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under which context fruitful discussions took place to shape this manuscript. All authors thank NanoTemper for constant support and discussion about effects and data analysis. Additional thanks go to Zuzana Gelova, Lorenzo Pichianti, Matthias Samwer and Sophie Woehrer for having samples that showed the interesting effects discussed in this manuscript.
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Special Issue: COST Action CA15126, MOBIEU: Between atom and cell.
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López-Méndez, B., Uebel, S., Lundgren, L.P. et al. Microscale Thermophoresis and additional effects measured in NanoTemper Monolith instruments. Eur Biophys J 50, 653–660 (2021). https://doi.org/10.1007/s00249-021-01529-1
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DOI: https://doi.org/10.1007/s00249-021-01529-1