3
APPLICATION NOTE
©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Results and Discussion
In order to validate the RED-NHS 2nd Generation kit
for MST interaction analysis, three different types of
interactions were quantified: protein-small molecule,
protein-protein and protein nucleotide. The three
target proteins were each labeled in two separate
batches, one using our conventional RED-NHS
labeling kit and one using the new RED-NHS 2nd
Generation kit. A er labeling, MST ligand-binding
experiments were run in a Monolith NT.115. Premium
capillaries were used as these are optimized to
prevent protein adsorption to the glass surface.
Additionally, experiments were set up with the latest
version of MO. Control so ware (v.1.6) with updated
analysis settings optimized for TRIC-sensitive dyes.
First, we looked at the interaction between p38
mitogen-activated protein kinase (p38α) and its
small inhibitory compound SB239063. This potent
p38α inhibitor was shown to exhibit an IC50 of 44 nM.
Figure 1 shows the MST traces as well as the binding
curves for experiments performed using both versions
of the RED-NHS labeling kit. Binding affinities of 65.5
± 17.1 nM for RED-NHS 2nd Generation and 84.0 ± 1.5
nM for RED-NHS were detected. While K
d
values are in
agreement, both response amplitude and S/N ratio
obtained with RED-NHS 2nd Generation-labeled p38α
are about twice as large as those obtained with RED-
NHS-labeled p38α (see also Table 1).
Figure 1: MST traces and binding curves for p38α-RED-NHS 2nd Generation (
•
) and p38α-RED-NHS (
•
) binding to SB239063. The obtained Kd values were
65.5 nM and 84.0 nM, respectively. Binding amplitudes and S/N ratios are listed for comparison in Table 1.
0 5 10 15 20
MST time [s] -on
1.0E-11 1.0E-10 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
Ligand Concentration [M]
0.75
0.80
0.85
0.90
0.95
1.00
Relative
Fluorescence
[-]
-10
-5
0
∆
FNorm
[
‰]
p38α-RED-NHS
p38α-RED-NHS 2nd Generation
