Hsp90, fluorescently labeled with NT647, was
used at a concentration of ~ 40 nM. The 17-
DMAG stock was dissolved in 50 % (v/v) ethanol
at a concentration of 500 µM. We used 50 µM
17-DMAG as the highest concentration for the
serial dilution. At this concentration the final
ethanol concentration still was 5 % (v/v). To make
sure that no buffer effects are observed the
dilution buffer was prepared accordingly making
sure that the final ethanol concentration was
~ 5 % (v/v) in all samples. After 1 min incubation
at room temperature the samples were loaded into
standard treated capillaries. Fig. 2 shows the initial
capillary scan, the shape of the MST curves and
the resulting binding curve. The initial capillary
scan already indicated slight unspecific binding of
the labeled Hsp90 to the capillary walls.
Subsequent analysis of the MST curves revealed
high fluctuations in the data.
Fig. 2 Binding of 17-DMAG to Hsp90 in standard treated
capillaries: initial capillary scan (upper), shape of the MST
curves (inset) and the resulting data points (n = 3 independent
measurements, lower). The initial capillary scan already
indicated slight unspecific binding of the labeled Hsp90 to the
capillary walls as does the high standard deviation of the data
points.
The presence of double peaks in the capillary
scan after the measurement in standard treated
capillaries (Fig. 3) clearly indicated unspecific
binding of the labeled Hsp90 to the capillary walls.
Fig. 3 Capillary scan after the measurement in standard
treated capillaries: the double peaks clearly indicate unspecific
binding of labeled Hsp90 to the capillary walls.
We therefore tested hydrophobic and hydrophilic
capillaries. Fig. 4 shows the initial scan, the MST
curves and the resulting binding curve for the
measurement in hydrophilic capillaries.
Fig. 4 Measurement of Hsp90 vs. 17-DMAG in hydrophilic
capillaries: initial capillary scan (upper), shape of the MST
curves (inset) and the resulting binding curve (n = 3
independent measurements, lower). The calculated K
d
of 0.503
± 0.099 µM is in good agreement with the published K
d
of 0.35
± 0.04 µM (Onuoha et al. 2007).
In comparison to the standard capillaries the
standard deviations of the data were small. Also
after the measurement no unspecific binding of
labeled Hsp90 was observed (Fig.5).