Fig. 5 Capillary scan after the measurement in hydrophilic
capillaries.
Fig. 5 Label-free measurement of Hsp90 vs.17-DMAG in thin
wall label-free capillaries. Pre-tests indicated that the 17-
DMAG itself shows no auto-fluorescence. From the resulting
binding curve (n = 2 measurements) a K
d
of 0.593 ± 0.387 µM
was calculated.
The calculated K
d
s from both measurements were
0.503 ± 0.099 µM and 0.593 ± 0.387 µM which
corresponds well to the published K
d
of 0.35 ±
0.04 µM (Onuoha et al., 2007).
Conclusion
The study provides another example that
MicroScale Thermophoresis is also capable of
measuring interactions of small molecules with
proteins. It also illustrates the high content
information of the measurement which allows
directly adjusting and optimizing the assay
conditions either by changing the type of
capillaries or by adjusting the buffer conditions.
Material and Methods
Assay conditions
For the experiment human Hsp90, purified as
previously described (McLaughlin et al., 2002),
was labeled with the Monolith NT™ Protein
Labeling Kit RED (Cat#L001) according to the
supplied labeling protocol. Labeled Hsp90 was
used at a concentration of ~ 40 nM. 17-DMAG
(Cambridge Bioscience) was titrated in 1:1
dilutions beginning at 50 µM, which still contained
5 % (v/v) ethanol. Samples were diluted in a
50 mM Tris-HCl, pH 7.4 containing 150 mM NaCl,
10 mM MgCl
2
and 0.05 (v/v) % Tween-20
supplemented with ethanol at a final concentration
of 5 % (v/v) to make sure that all samples
contained the same ethanol concentration. For the
measurement the samples were filled into
hydrophilic capillaries (Cat#K004) or thin wall LF
capillaries.
Instrumentation
The measurements were performed on a
NanoTemper Monolith NT.115 instrument and the
Monolith NT-LabelFree.
The measurement was performed at 40 % LED
and 40 % MST power, Laser-On time was 30 sec,
Laser-Off time 5 sec.
Label-free measurements were done on the
Monolith NT-LabelFree in thin wall LF capillaries
at 20 % MST power, Laser-On time was 30 sec,
Laser-Off time 5 sec.
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© 2011 NanoTemper Technologies GmbH