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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Protein unfolding and denaturation is one of the major
causes for protein loss-of-function, and must therefore
be carefully controlled in drug discovery and drug
development. For instance, therapeutic antibodies must
maintain their functionality when stored under different
conditions, and must also tolerate moderate physical
stress (e.g. shaking) as well as changes in temperature.
Moreover, many potential drug targets such as integral
membrane proteins, cell surface receptors or kinases,
must maintain their functionality during cost-intensive
screening campaigns, and might react very differently to
physical and thermal stress.
Figure 1: Example of a typical thermal unfolding curve by nanoDSF.
The fluorescence ratio F350/F330 can be expressed as % unfolded if the
fluorescence ratios of the folded and unfolded states are known.
50 60 70 80
Temperature (°C)
0.4
0.5
0.6
0.7
0.8
0.9
F350/F330
0
20
40
60
80
100
%
unfolded
Here we show that the Prometheus NT.48 allows for
a precise determination of the fraction of unfolded
protein in just a few µl of protein formulations within
seconds.
Prometheus measures thermal unfolding profiles of
proteins by detecting even minute changes in the
emission properties of the amino acid tryptophan
upon unfolding. By following the shi in the
fluorescence emission wavelength, an unfolding
curve can be generated (Figure 1). Importantly, the
specific fluorescence ratios (F350/F330) of the folded
and unfolded states can be directly correlated to the
respective fraction of unfolded protein at any point
during the unfolding process (Figure 1). This also
means that a single fluorescence scan of a protein
sample contains information about the overall amount
of unfolded protein in the protein sample, once the
respective F350/F330 values of the folded and unfolded
state are known. This information is valuable for protein
quality control, either for evaluating long-term stabilities
of biologicals or the effects of physical or thermal stress
on protein stability.
Prometheus uses a capillary "dip-and-read" format,
meaning that the capillaries can be directly loaded
by dipping into a protein solution without additional
pipetting steps. The measurement of the F350/F330
