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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Conclusion
The presented data demonstrate that the Prometheus
NT.48 can be employed to quickly detect and quantify
unfolded proteins for quality control purposes with
unmatched speed, at the same time offering unique
ease of use. The presented quality control experiments
can be performed by filling capillaries directly from
stock solutions without laborious sample preparation
or loading. F350/F330 values of up to 48 samples are
then recorded in parallel using a one-button routine,
providing stability data within seconds.
Material and Methods
Protein preparation
IgG unfolding standards were created by incubating
mouse IgG from serum (Sigma Aldrich) at a
concentration of 2 mg/mL in PBS, pH 6.2 at 75 °C with
shaking for 3 h. The fully unfolded IgG was then cooled
on ice and mixed with folded IgG in the same buffer to
create formulations of 2 mg/mL IgG with the fractions of
unfolded IgG shown in Figure 2.
HiTehA long-term storage experiments were performed
by incubating aliquots of HiTeHA at a concentration of
2 mg /mL in 50 mM Tris, 200 mM NaCl, 0.02 % DDM, pH 8
at 4 °C and at RT. For each experiment, 5 Prometheus
capillaries per condition were filled with 10 µl of sample,
respectively, and subjected to a thermal ramp from
25 °C to 90 °C with a heating rate of 1 °C/min.
The MEK1 forced degradation screen was performed
with 2.9 µM MEK1 in 20 mM HEPES pH 7.4, 300 mM NaCl,
2 mM DTT. For each treatment, aliquots of 50 µl were
used and subjected to the treatments described in the
results section. Samples from each condition were filled
into 3 Prometheus capillaries, respectively, and subject
to a thermal ramp from 25 °C to 90 °C with a heating rate
of 1 °C/min.
