4
ASSAY DEVELOPMENT TECHNICAL NOTE
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Conclusions
Using the advanced Binding Check feature in the MO.Control 2 so ware included with
Monolith, 6 buffer conditions were tested in parallel to study the molecular interaction
between Hsp90 and ADP. In less than 15 minutes, signal-to-noise ratios for the 6
conditions were measured and automatically compared. Significant differences were
observed between buffers and ranged from no interaction to aggregation to strong
binding. The buffer condition that showed a strong interaction was used in a subsequent
affinity quantification experiment with the Binding Affinity module. Results showed a
K
d
that was in agreement with published data, demonstrating the quality of the Binding
Check analysis.
The importance of determining optimal assay buffer conditions cannot be understated
since assay buffers can alter protein structure and function. This applies to MST and
any other biophysical methods used. For MST experiments, this straightforward Binding
Check experiment can be used to speed up assay development time by determining
optimal buffer conditions quickly and conclusively.
and into the Monolith instrument. MST measurement was carried out at 25 °C using 30%
LED and high MST power. Acquired data sets were analyzed with MO.Affinity Analysis
3 so ware at 10 seconds MST on-time, resulting in a K
d
of 18 ± 3 µM, which is in good
agreement with published affinity data for Hsp90 and ADP
6
. Figure 3 shows the MST traces
(le panel) and the binding curve (right panel) for the performed experiment.
0 5 10 15 20
0.80
0.85
0.90
0.95
1.00
0.1 1 10 100
10 3
10 4
-30
-25
-20
-15
-10
-5
0
Figure 3: MST traces (le panel) and dose-response curve (right panel) for ADP titrated with labeled Hsp90 at high MST power. An MST on-time of 10 sec was used for
analysis, and a K
d
value of 18 ± 3 µM was obtained (n = 2 independent measurements, error bars represent the standard deviation).
