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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
denaturation of proteins within seconds. In summary,
our results demonstrate that the Prometheus is
exceptionally well suited for a rapid, precise and cost-
effective characterization of protein stability, both in
academic and industrial settings. Its flexibility and
speed make it a valuable tool for a plethora of different
experimental approaches, ranging from in-depth
characterization of protein folding to high-throughput
screening projects.
Material and Methods
Sample preparation
α-amylase from pig (pig pancreatic α-amylase, PPA,
Roche) and α-amylase from Aspergillus oryzae, TAKA,
Sigma) were dissolved in 30 mM HEPES, 50 mM NaCl,
2 mM CaCl
2
, pH 7.4 at concentrations of 10 mg/mL. Final
concentrations in thermal unfolding experiments were
10 µM. In order to remove residual traces of ammonium
sulfate or other contaminants, a buffer exchange
using buffer exchange spin columns was performed
(NanoTemper Technologies). For the determination of
the Ca
2+
-dependence of α-amylase stability, a second
buffer exchange into buffer lacking CaCl
2
but including
5 mM EDTA was performed.
For the formulation screen, the proteins were
transferred into 20 mM Na citrate buffer, pH 5.9, with the
respective concentrations of sucrose, sorbitol, trehalose
or glycerol.
Thermal unfolding experiments
For thermal unfolding experiments, the proteins were
diluted to a final concentration of 10 µM. For each
condition, 10 µl of sample per capillary were prepared.
The samples were loaded into Prometheus capillaries
(NanoTemper Technologies) and experiments
were carried out using the Prometheus NT.48. The
temperature ramp was set to an increase of 1 °C/min
in a range from 20 °C to 90 °C. Protein unfolding was
measured by detecting the temperature-dependent
change in tryptophan fluorescence at emission
wavelengths of 330 and 350 nm.
