3
TECHNICAL NOTE
©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
- 1 0 1 2 3 4 5
T i m e [ s ]
0 . 9 4
0 . 9 5
0 . 9 6
0 . 9 7
0 . 9 8
0 . 9 9
1
- 1 0 1 2 3 4 5
T i m e [ s ]
0 . 9 4
0 . 9 5
0 . 9 6
0 . 9 7
0 . 9 8
0 . 9 9
1
- 1 0 1 2 3 4 5
T i m e [ s ]
0 . 9 4
0 . 9 5
0 . 9 6
0 . 9 7
0 . 9 8
0 . 9 9
1
R
e
l
a
t
i
v
e
F
l
u
o
r
e
s
c
e
n
c
e
[
-
]
A B C
Figure 1: TRIC-traces (fluorescence intensity change measured over time) for G9a in absence (green) and presence (purple) of SAM. The areas highlighted between two
traces represent Ligand Area (A, purple) or the area between the two ligand replicates, Reference Area (B, green) or the area between the two reference repeats and one
of four possible Signal Areas (C, grey). As there are two reference replicates and two ligand replicates, there are four possible areas between a reference replicate and a
ligand replicate.
and positive control or ligand. The larger the Signal Area of the tested positive control is,
the more sensitive the assay will be to binders with a lower signal amplitude. The Signal
Quality value (SQ) for the different buffer conditions is then calculated with the following
formula:
From the formula, one can infer that the SQ is a measure of both sensitivity (because
it includes the signal areas) and reproducibility (by including the reference and ligand
areas). The DI.Control so ware automatically calculates the SQ for all 96 conditions and
displays it as a heatmap (Figure 2, right panel), where darker shades of blue point to a
buffer formulation that results in a higher Signal Quality. In our assay, the lightest color
corresponds to an SQ of 0 and the darkest or maximum value measured to an SQ of 18.63.
With the systematic arrangement of conditions on the plate, the positive or negative
SQ =
Signal Area
(Reference Area + Ligand Area)/2
