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TECHNICAL NOTE
Prometheus and NanoTemper are registered trademarks of NanoTemper Technologies GmbH, Munich, Germany © 2019
homogeneity of results across all 48 sample positions
(capillaries) within one instrument and across 10
different Prometheus instruments. This was done by
investigating thermal unfolding profiles of a reference
protein (streptavidin) at 1 mg/mL.
Calculated T
m
values were used to assess temperature
control precision. The initial ratio – the ratio of emission
intensity at 350 nm and 330 nm wavelength (F350/F330
ratio) at the beginning of the measurement – was used
to assess precision of fluorescence detection. The initial
ratio is particularly well suited for this purpose, since it
Repeatability
SD RSD (CV)
Average 48 sample initial ratio variation 0.008 0.4 %
48 sample T
m
variation (@75 °C) 0.1 °C 0.07 %
Intermediate precision
SD RSD (CV)
Initial ratio variation between 10 instruments 0.02 2.2 %
T
m
variation between 10 instruments (@75 °C) 0.33 °C 0.4 %
Table 1: Measurement precision across 10 instruments summarizing repeatability and intermediate precision results.
also reflects the positioning of capillaries relative to the
optics assembly. Even small irregularities in the optics
or changes in the distance of optics to the capillaries
along the sample tray would result in easily observable
trends in the initial ratio.
Figure 1 shows the overlay of the ratio F350/F330 signal
(top) and its first derivative (bottom) for all 48 samples
during a single measurement. The figure demonstrates
the strong homogeneity across the entire sample tray of
an instrument, which is very important to ensure intra-
assay comparability of sample data.
