3
TECHNICAL NOTE
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detector of the Monolith Pico instrument at 10% LED and medium MST power. Data
was analyzed a er 5 seconds MST on-time using the MO.Affinity Analysis 3 so ware.
The resulting dose-response curves were fitted to a one-site binding model to extract
K
d
values. Fraction-bound results are shown in Figure 2, together with obtained K
d
s,
which are in good agreement with published values
1,2
. Using the RED detector not only
allowed precise quantification of the lower nM K
d
of Acetazolamide, but also required
much less protein sample compared to the protein concentrations needed for label-free
experiments.
0.01 0.1 1 1 0 1 00
1 0 3
1 0 4 1 0 5 1 0 6
Ligand Concentration [nM]
0.0
0.5
1.0
Fraction
Bound
[-]
Figure 2: Precise K
d
determination
of a broad range of affinities
using the RED detection of
Monolith Pico. Dose-response
curves for Acetazolamide (dark
blue), Furosemides (light blue),
and Benzenesulfonamide (purple)
titrated with labeled CAII at medium
MST power. An MST on-time of
5 seconds was used for analysis,
and K
d
values of 13 ± 2 nM, 411 ±
80 nM and 2 ± 0.4 µM, respectively
were obtained (n = 3 independent
measurements, error bars represent
the standard deviation).
Conclusions
The combination of LabelFree and RED detectors in the Monolith Pico instrument
provides scientists with the ability to characterize almost any type of interaction. Where
binding affinity quantification under native conditions is preferred, label-free MST can
be performed. In other circumstances, measuring a labeled target is desired and the RED
channel is useful for measuring any interaction, even the most challenging ones — those
involving ternary complexes or interactions in cell lysate or serum — while using very
little sample. Having both detectors in one instrument provides the highest versatility
and flexibility of all Monolith instruments, enabling scientists to freely choose the most
appropriate approach for their project.
