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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
the use of Prometheus capillaries offers an even
higher precision for UV-fluorescence detection than
high-performance quartz-cuvettes, with the additional
benefit of low sample consumption, high-throughput
and high versatility. Moreover, the capillary-based
approach prevents cross-contamination, and no
laborious and time-consuming cleaning steps are
required. High scanning rates and thus high data density
moreover allow for a robust analysis of melting curves,
and also enables a precise determination of unfolding
onsets.
In addition, the direct detection of tryptophan
fluorescence to monitor protein unfolding has several
benefits compared to other methods routinely used to
monitor thermal unfolding, such as differential scanning
fluorimetry (DSF) or thermofluor assays. These assays
use external fluorophores which bind to hydrophobic
patches of the protein usually buried in the core of the
protein. Upon unfolding, these patches get exposed
and the fluorophore attaches, resulting in an increase
in fluorescence. These assays, however, are not suited
for a detailed analysis of folding thermodynamics,
since they interfere with folding-unfolding equilibria
by directly interacting with the proteins. Moreover, the
external fluorophores are incompatible with a number
of buffers (e.g. including detergents) or protein types,
such as membrane proteins. Lastly, although DSF
is routinely used in primary screenings in the drug
discovery process, external fluorophores can interact
with compounds or block binding sites and produce
false-negative as well as false-positive results.
In addition to its capabilities in monitoring thermal
unfolding of a large number of samples in parallel,
the Prometheus can also be used to analyze chemical
Figure 4: Ca
2+
effects on amylase stability. Removal of Ca
2+
ions results in a marked destabilization of both amylase isoforms, as indicated by the shi of T
m
towards
lower values.
Pig pancreatic α-amylase PPA
Aspergillus orzae α-amylase TAKA
Temp (°C) Temp (°C)
