5
APPLICATION NOTE
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freeze-thaw cycles. Again, the fluorescence ratio F350/
F330 served as an indicator for the fraction of unfolded
protein (Figure 4).
Incubation of MEK1 for 1 h on ice or at RT resulted in no
significant difference in the F350/F330 ratio, showing
that the protein can be handled at RT over this time
period without risking protein denaturation. This
finding is in agreement with the previously performed
MST-based fragment screening campaign, in which
the MEK1-nucleotide interaction was shown to be
stable for > 12 h at RT [2]. In contrast, incubation at an
elevated temperature of 40 °C for 1 h, which is above
the unfolding onset temperature, already resulted in
a fraction of unfolded protein of ~65 %. Incubation
of the protein solution for 15 minutes at 60 °C led to
a complete unfolding of the protein, as expected,
indicated by the absence of an unfolding transition in
the thermal unfolding curve. Interestingly, the protein
was rather resistant towards mechanical stress, as
vortexing for 1 minute at maximum rpm resulted in only
a small fraction of unfolded protein (< 5 %). In contrast,
repeated freeze-thaw cycles strongly denatured the
protein, leading to > 75 % unfolded protein (Figure 4).
Thus, this set of experiments quickly revealed that MEK1
is stable at ambient temperature and rather insensitive
against mechanical stress such as vortexing, whereas
freeze-thaw cycles and elevated temperatures promote
the denaturation of the protein and should be avoided.
30 40 50 60 70 80 90
0.55
0.60
0.65
0.70
0.75
0.80
0.85
Temperature (°C)
F350/F330
10x freeze-thaw
1 min vortex
15 min 60°C
1h 40°C
1h 4°C
1h RT
1h
RT
1h
4°C
1h
40°C
15
min
60°C
1
min
vortex
10x
freeze-thaw
100
75
50
25
0
%
unfolded
MEK1-kinase forced degradation
Figure 4: Forced-degradation stress-test on MEK1. MEK1 protein was subject to
the indicated stresses, and the fraction of unfolded protein was calculated based
on the F350/F330 ratio at 25 °C. Error bars are s.d. from three measurements.
