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The effect of storage-related stress on the outcome
of biophysical experiments was then evaluated by
measuring the interaction of the p38 samples with
the small molecule inhibitor SB239063 by MicroScale
Thermophoresis (MST) (Figure 1B). Results of
the MST analysis confirmed that the presence of
unfolded protein in the sample directly influences
T
i
(°C) Initial Ratio △ Ratio
MST Kd
(nM)
MST amplitude
(F
norm
(
0
/
00
))
MST
signal-to-noise
Fresh 53.1 0.725 0.193 59 28.5 25.7
Freeze-thaw cycles 53.2 0.739 0.176 64 21.6 7.4
50% denatured 53.5 0.812 0.088 68 12.8 9.5
Fully denatured n.a. 0.852 0.025 n.a. 0 n.a.
Table 1: Summary of Tycho NT.6 and Monolith analysis of p38 kinase under different storage conditions
assay outcome. The MST binding amplitude
decreased with the presence of increasing amounts
of unfolded protein and the overall assay quality
was lower, as indicated by greatly reduced signal-to-
noise values (Table 1). Fully denatured p38 did not
show an interaction.
Conclusions
We show that the Tycho NT.6 system can be used to
quickly evaluate different storage conditions, such
as freezing strategies, to provide a better insight on
the quality of the proteins being tested. The results
demonstrate that poor sample quality and the
presence of denatured protein can directly influence
assay performance and the experimental outcome.
The Tycho NT.6 system is well-suited to swi ly
validate protein quality in as short as 3 minutes, prior
to performing further biophysical analysis which can
be time-consuming and costly to run.