APPLICATION NOTE
5
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Non-binder
77.03%
Potential Hit
9.2%
Not Reproducible
6.83%
Inhomogeneous ΔFnorm
2.01%
Scan Anomaly
Auto-fluorescence
Agreggation
Hit
3.53%
88 Hit
Aggregation 9
Auto-fluorescence 3
Non-binder 1981
Potential Hit 229
Non Reproducible 170
Inhomogeneous ΔFnorm 50
Scan Anomaly 23
Total 2490
Figure 2: Single-dose screen summary. Fragments are sorted into categories based on sorting algorithm in DI.Screening
Analysis so ware.
an expected outcome from using a non-specific library. Eighty-eight fragments
(3.5%) were identified as hits. They ranged from ~100 Da to ~350 Da in molecular
weight and from ~90 to ~400 in molecule complexity according to the Bertz/
Hendrickson/Ihlenfeldt formula
6.7
. From the list of fragment hits, a few were
selected for the second step, affinity screening, involving titration experiments to
determine specificity and affinity (K
d
).
For the affinity screening, a 12-point, 0.5-fold dilution series of the selected
fragments were prepared, starting at 1 mM. Then, labeled G9a was added at 5
nM final concentration to each of the 12 ligand dilutions. The dose-dependent
response curves, resulting from Fnorm analysis a er a 2.5 second IR-laser
on-time, were approximated with a fit model that describes the law of mass
action. Fragments were characterized by different binding affinities and signal
amplitudes.
Table 1 shows four fragments and the positive control SAM. The figures
demonstrate good correlation between the single-dose and affinity screens.
