Abstract
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.
Highlights
The NanoTemper Monolith was introduced as a commercial instrument in 2011, following the accomplishments of academic studies in the years 2006–2010 (Duhr and Braun 2006; Jerabek-Willemsen 2011; Jerabek-Willemsen JerabekWillemsen 2014)
31 NT.115 and 9 NT.Pico instruments were used in the benchmark study
Absolute fluorescence counts per LED power as well as the bleaching effect per second and LED power showed significant differences for NT.115 instrumentation prior to 2013 (Fig. 3a, b) but not in NT.Pico instruments which can be explained by a change in hardware by NanoTemper for more recent instruments
Summary
The NanoTemper Monolith was introduced as a commercial instrument in 2011, following the accomplishments of academic studies in the years 2006–2010 (Duhr and Braun 2006; Jerabek-Willemsen 2011; Jerabek-Willemsen JerabekWillemsen 2014). Despite successive generations of instruments sharing the same name (Monolith NT.115), changes in the hardware, software and best practices in data analysis have occurred over the last 10 years. European Biophysics Journal (2021) 50:411–427 the software and data analysis practices independently of each other. To achieve this goal, all sample stocks were centrally prepared. An exhaustive standard operating procedure (SOP) for the sample preparation and settings for the measurement was prepared, to be followed by each participant of the benchmark (see supplementary material 4). Possible standards and labeling procedures as well as instrument settings were tested in a small-scale benchmark within the ARBRE-MOBIEU working group prior to the start of this wider benchmark study. To include as many participants as possible and according to our information that most instruments sold contained the red channel, a red dye was chosen and only instruments with red filter sets were eligible
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