4
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Binding assays
Next we tested whether RED-tris-NTA labeled
His-tagged proteins can be used for interaction
quantification by MST. We first tested protein-small
molecule interactions, using purified His6-IDH R132H
and His6-p38α. Figure 3 shows that clear binding
events with excellent signal-to-noise ratios could be
measured by MST for both systems. The affinity of
PD169316 for p38α was 124 ± 8 nM and the affinity
of C35 for IDH R132H was 100 ± 14 nM, which is in
excellent agreement with published data (Nordin,
Jungnelius et al., 2005, Rohle, Popovici-Muller et al.,
2013). Determined K
d
values for the PD169316-p38α
interaction were also comparable to data obtained
with traditional RED-NHS labeling. Interestingly, the
IDH1 R132H-C35 interaction yielded much better
dose-response curves than approaches with standard
RED-NHS labeling, suggesting that His-tag labeling by
Tris-NTA is the method of choice for labeling of fragile
proteins which are sensitive towards the respective
labeling buffer conditions or covalent modifications.
β-mercapto-ethanol 1 mM
GSH 10 mM
GTP, GDP 1 mM
AMP, ADP, ATP 5 mM
Glycerol 10 %
His- tagged ligand None
Table 1: List of common buffer components and their maximum allowed
concentration
Overall, the RED-tris-NTA labeling procedure
exhibits low sensitivity towards an array of different
components, which are o en used in storage buffers.
Even competitors like histidine and imidazole
only interfere with the labeling at concentration
higher than 1 mM. Reducing agents, which are not
tolerated during standard NHS labeling, also do not
significantly interfere with RED-tris-NTA labeling.
This means that in most cases direct labeling of the
protein in its storage buffer is possible, and no buffer
exchange is required prior to labeling with RED-
tris-NTA. Interestingly, we in addition observed that
RED-tris-NTA dye exhibits a much lower degree of
photobleaching when compared to the standard RED
dye, which is likely caused by the presence of nickel(II)
in the complex (Glembockyte, Lincoln et al., 2015).
