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ASSAY DEVELOPMENT TECHNICAL NOTE
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The so ware not only guides the user step by step
through the Binding Check experiment, assisting
with sample preparation and selection of the right
measurement parameters, but it also provides detailed
interpretation of the data.
This buffer was then used to measure binding affinity
of Hsp90 and ADP, which gave a K
d
that matched
published data, thereby demonstrating the strength
of the Binding Check module for fast and high-quality
buffer optimization.
Results
To prepare for the binding experiments, the target
protein Hsp90 was fluorescently labeled using the RED-
NHS 2nd Generation dye according to the instructions in
the labeling kit (Cat# MO-LO11).
Binding of ADP and labeled Hsp90 was tested in 6
different assay buffers altogether in one experiment.
For each buffer condition, target alone (unbound) and
target in complex with ligand (bound) were analyzed
in duplicate. For each sample, 10 µL of 40 nM target
was mixed with either 10 µL of buffer (unbound), or
with 10 µL of 1.6 mM ligand (bound). Samples were
transferred to premium coated capillaries
(Cat# MO-K025) and placed into the 24 positions on the
sample tray. Binding was measured using the Binding
Check module in the MO.Control 2 so ware of the
Monolith instrument. Data was recorded at 30% LED
and high MST power at 25 °C.
Figure 2 shows the results of the Binding Check
experiment for Tris buffer (50 mM Tris pH 7.4, 150 mM
NaCl, 10 mM MgCl
2
), supplemented with 0.05%
Tween-20, which the so ware automatically identified
as the buffer with the highest signal-to-noise ratio.
Table 1 lists the results of the Binding Check experiment
for all tested buffers.
Figure 1: Monolith instrument equipped with the MO.Control 2
so ware for rapid assay development.
It calculates the signal-to-noise ratio for each condition
tested and identifies the best buffer that should be used
for any further experiments.
In order to test the capability of the Binding Check
module, 6 different buffer conditions were compared for
the interaction analysis of heat shock protein 90 (Hsp90)
and Adenosine-5'-diphosphate (ADP).
In less than 15 minutes, the
MO.Control 2 so ware identified the
buffer with the best signal-to-noise
ratio for this interaction
