5
APPLICATION NOTE
©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Finally, the interaction between human heat shock
protein 90 (Hsp90) and adenosine diphosphate
(ADP) was tested using both labeling kits. Hsp90
is a dimeric protein with ATPase activity, crucial
for the cellular heat shock response and o en
considered a potential target for the treatment of
neurodegenerative diseases and cancer. MST traces
and dose response curves are shown in Figure 3.
When HSP90 was labeled with the RED-NHS kit, a K
d
determination was not possible due to low signal to
noise levels. In contrast, a K
d
was identified for the
interaction between Red-NHS 2nd Generation-labeled
Hsp90 and ADP (65.8 ± 12.0 µM). Also for the Hsp90-
ADP interaction, binding amplitude and S/N ratio are
significantly larger for protein labeled with RED-NHS
2nd Generation – in this case, by factors of 2.5 and 5
respectively (see also Table 1).
An additional advantage is that data evaluation at
very early MST-on times already yields high-quality
results, potentially allowing for shorter measurement
durations. This is of high relevance in drug discovery
workflows, where time-consuming screenings may
be shortened significantly. High S/N ratios could be
obtained even a er the shortest possible MST-on
times (1.5 s), while with the conventional RED-NHS
dye, sufficient S/N levels were usually reached at later
times (in the case of p38α-SB239063) or not at all (in
the case of TEM1-BLIP and Hsp90-ADP). MO.Control
so ware v1.6 automatically evaluates data by finding
the earliest possible MST-on time, ensuring that
high-quality data is generated swi ly and without
additional user intervention.
Amplitude S/N ratio
Labeling kit used to
label target protein
RED-NHS
Interaction
p38α
vs. SB239063
TEM1 vs. BLIP
Hsp90 vs. ADP
RED-NHS
2nd Generation
5.9
1.9
0.9
10.9
11.9
2.5
12.9
6
3
24.5
35.4
15.8
RED-NHS
RED-NHS
2nd Generation
Table 1: Comparison of binding amplitudes and S/N ratios. RED-NHS 2nd Generation performs significantly better on both counts.
