APPLICATION NOTE
8
©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
-5
0
5
10
15
20
Fnorm
[‰]
0.92
0.96
1.00
Relative
Fluorescence
[-]
-1 0 1 2 3 4 5
Time [s]
950
960
970
980
990
1000
Fnorm
[‰]
1x10
2
1x10
3
1x10
4
1x10
5
1x10
6
1x10
7
Ligand Concentration
0.92
0.94
0.96
0.98
1.00
Relative
Fluorescence
[-]
-1 0 1 2 3 4 5
Time [s]
Fragment D85021
Table 1: Column on the le shows the scatter plot with the ΔFnorm response (A) and TRIC signal plotted as the relative fluorescence vs. time
(B) from the single-dose screen. Dots and traces in green correspond to the signal measured from the reference (DMSO), dots and traces in
blue represent signal measured from the positive control (SAM) and fragments. Column on the right represents the TRIC signal (C) and dose
response curves resulting from the titrations of selected fragment molecules and SAM against G9a (D). Dots and traces in grey correspond
to data that was identified by the so ware to be biased by aggregation. Concentrations in the dose response plots are indicated in nM.
(A) (C)
(B) (D)
Ligand K
d
(mM)
Amplitude,
ΔFnorm (‰) S/N ratio
SAM 0.013 22.5 38.0
A12935 0.656 12.5 37.7
C18598* >0.299 15.1 41.7
A25749 0.386 5.8 28.2
D85021* >2.270 50.2 136.8
Table 2: K
d
values, amplitudes and S/N ratios for positive control SAM and the four fragments selected for affinity
screening. *Saturation of binding curve is insufficient to determine a more precise estimation of the K
d
value.
Overall, affinities were in the higher micromolar range, as expected from fragment molecules (Table 2).
Importantly, the measured dissociation constant for SAM, K
d
=13.3 µM, was in good agreement with published
values derived from ITC data
8
.
Single-dose screen
(A) ΔFnorm
(B) TRIC trace
Affinity screen
(C) Fnorm dose response curve
(D) TRIC trace
Fragment
