3
ASSAY DEVELOPMENT TECHNICAL NOTE
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Only Tris buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl
2
), supplemented with
0.05% Tween-20 showed a high enough signal-to-noise ratio to conclude binding. All
other buffers either led to sample aggregation or showed an insufficient signal-to-noise
ratio.
0.82
0.84
0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
910.0
910.5
911.0
911.5
912.0
912.5
913.0
-2 0 2 4 6 8 10 12 14 16 18 20 22
Figure 2: Binding Check results
for the interaction of ADP and
Hsp90 in 50 mM Tris buffer pH 7.4,
supplemented with 150 mM NaCl,
10 mM MgCl
2
and 0.05% Tween-20.
MST traces (le panel) and Fnorm
values of Hsp90 alone (target) and in
complex with ADP (complex) (right
panel). An MST on-time of 1.5 sec
was used for analysis. A signal-to-
noise ratio of 13.3 and a binding
amplitude of 2.8 was obtained (n = 2
independent measurements).
Table 1: Summary of results from
Binding Check experiment for the
interaction of Hsp90 with ADP.
Buffer # Buffer composition Buffer Check result
1
50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl
2
Buffer #1, supplemented with 0.05% Tween-20
20 mM HEPES pH 7.4, 150 mM NaCl
Buffer #3, supplemented with 0.05% Tween-20
Phosphate buffered saline (PBS) pH 7.4
Buffer #5, supplemented with 0.05% Tween-20
S/N ratio too low to conclude binding*
Binding detected
S/N ratio too low to conclude binding*
S/N ratio large enoughto conclude binding*
S/N ratio too low to conclude binding
Response amplitude too slow
2
3
4
5
6
* sample aggregation
Subsequent affinity quantification of Hsp90 and ADP in Tris buffer with 0.05% Tween-20
was performed using the Binding Affinity module in the MO.Control 2 so ware. For
this, a 12-point 1:1 serial dilution of the ligand ADP was prepared starting at a highest
final assay concentration of 1.13 mM. Hsp90 was added to all dilution steps with a final
concentration of 20 nM. Samples were prepared in duplicate and both dilution series
were transferred to premium coated capillaries (Cat# MO-K025) and loaded onto the tray
