3
TECHNICAL NOTE
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Tween as assay buffer. Triplicate measurements were
performed with Monolith NT.115 in Premium Capillaries
(Cat# MO-K025) at medium MST and 40% LED power.
The system was operated with the MO.Control so ware
v 1.6. Additionally, acquired data sets were analyzed by
MO.Affinity Analysis so ware v 2.3 at 2.5 sec MST on-
time.
Figure 2 shows the MST traces (le panel) as well as the
binding curves (right panel) for experiments performed
using positive and negative control RNA molecules and
labeled RNA-binding protein. A K
d
of 151 ± 9 nM was
obtained for the positive control RNA. As expected, no
binding was detected for the negative control RNA.
0 5 10 15 20
MST experiment time [s]
0.80
0.85
0.90
0.95
1.00
Relative
Fluorescence
[-]
0.01 0.1 1 10 100
10
3
10
4
10
5
Ligand Concentration [nM]
0
5
10
15
Δ
FNorm
[‰]
pos. control RNA
neg. control RNA
Conclusions
NanoTemper Technologies SNAP-Tag® Labeling Kit RED 2nd Generation provides a site-specific covalent labeling of puri-
fied proteins for MST and TRIC experiments. This labeling strategy guarantees a highly specific and controlled conjugation
of fluorophores to any SNAP-tagged protein since SNAP-Tag® substrates are inert to any other protein structure.
Figure 2: MST traces (le panel) and dose-response curves (right panel) for RNA (negative control in dark blue, positive control in light blue) titrated against labeled
RNA-binding protein at medium MST power. An MST on-time of 2.5 sec was used for analysis, and a K
d
value of 151 ± 9 nM was obtained (n = 3 independent measure-
ments, error bars represent the standard deviation).
