4
APPLICATION NOTE
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for protein labeled with RED-NHS 2nd Generation
– in this case, by about a factor of 6 (see also Table
1). A better S/N ratio for this particular interaction
could only be achieved at high MST power (data not
shown). It is worth noting that in general, RED-NHS
2nd Generation will yield better data at lower MST
power settings, which is usually the preferred and
recommended approach.
The protein-protein interaction between labeled
ß-lactamase TEM1 (a point mutant variant, P107A)
and ß-lactamase inhibitory protein (BLIP) was
examined next. MST traces and binding curves are
shown in Figure 2. Both labeling approaches yield
similar K
d
s (25.8 ± 6.0 nM using the RED-NHS 2nd
Generation and 23.2 ± 7.7 nM using the RED-NHS kit).
Again, binding amplitude and S/N ratio are boosted
0 5 10 15 20
MST time [s] -on
0.80
0.85
0.90
0.95
1.00
Relative
Fluorescence
[-]
1.0E-12 1.0E-11 1.0E-10 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
Ligand Concentration [M]
-10
-5
0
∆
FNorm
[‰]
TEM1-RED-NHS
TEM1-RED-NHS 2nd Generation
Figure 2: MST traces and dose response curves for TEM1-RED-NHS 2nd Generation (
•
) and TEM1-RED-NHS (
•
) binding to BLIP. The obtained K
d
values were
25.8 nM and 23.2 nM, respectively. Binding amplitudes and S/N ratios are listed for comparison in Table 1.
1.0E-08 1.0E-07 1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02
-3
-2
-1
0
1
∆
FNorm
[‰]
Ligand Concentration [M]
Hsp90-RED-NHS
Hsp90-RED-NHS 2nd Generation
MST time [s] -on
0 5 10 15 20
0.80
0.85
0.90
0.95
1.00
Relative
Fluoresc
ence
[-]
Figure 3: MST traces and dose response curves for Hsp90-RED-NHS 2nd Generation (
•
) and Hsp90-RED-NHS (
•
) binding to ADP. The obtained K
d
value for
Hsp90-RED-NHS 2nd Generation was 65.8 mM. K
d
determination was not possible with NT-647 due to low signal to noise levels. Binding amplitudes and S/N
ratios are listed for comparison in Table 1.
