SYPRO Orange using appropriate optics and a detector gain
(see Note 13) that ensures that the dynamic range of the
instrument is not exceeded at higher temperatures (i.e., do
not allow the fluorescence intensity to exceed the saturating
value for the detector).
7. Fluorescence intensity data should be acquired at 1
C intervals, or more frequently if greater reproducibility and precision
of thermal denaturation midpoint is desired (see Note 14).
3.2.2 Thermal Shift
Assay Data Analysis
and Typical Results
1. The thermal denaturation experiment should yield an approximately sigmoid denaturation curve with a steeply sloping posttransition baseline, similar to that shown in Fig. 1a.
2. Take the first derivative of the fluorescence intensity data. Most
thermocycler software will perform this automatically as part of
the standard analysis (see Note 15). The first derivative of the
raw data in Fig. 1a is shown in Fig. 1b.
3. Find the maximum of the first derivative, which corresponds
(approximately) to the apparent midpoint of thermal denaturation (T m ). Most thermocycler software will do this automatically, with an adjustable threshold so that noisy baseline regions
can be excluded from the search for maxima.
4. Average the T m values from replicate measurements and calculate the ΔT m according to ΔT m ¼ T m
HEWL+NAG3
À T m
HEWL
.
In five replicates of this experiment, each consisting of triplicate
samples for each condition, the mean T m for HEWL was
76.5 Æ 0.2
C and the mean T m for HEWL + 1 mM NAG3 was
79.6 Æ 0.3
C, and the mean ΔT m was 3.2 Æ 0.4
C (see Note 16).
Thus, there is clear evidence of binding of NAG3 to HEWL under
these conditions, indicated by a significant stabilization of HEWL
in the presence of 1 mM NAG3.
a
b
40
60
Temperature (°C)
Temperature (°C)
80
0
20
40
60
Fluorescence Intensity
(Arbitrary Units)
40
60
80
-5
0
5
10
First Derivative
Fig. 1 DSF raw fluorescence intensity data (a) and first derivative (b) for HEWL in the presence (red) and
absence (blue) of 1 mM NAG3
56
Xiaochun Li-Blatter et al.
(see Note 13) that ensures that the dynamic range of the
instrument is not exceeded at higher temperatures (i.e., do
not allow the fluorescence intensity to exceed the saturating
value for the detector).
7. Fluorescence intensity data should be acquired at 1
C intervals, or more frequently if greater reproducibility and precision
of thermal denaturation midpoint is desired (see Note 14).
3.2.2 Thermal Shift
Assay Data Analysis
and Typical Results
1. The thermal denaturation experiment should yield an approximately sigmoid denaturation curve with a steeply sloping posttransition baseline, similar to that shown in Fig. 1a.
2. Take the first derivative of the fluorescence intensity data. Most
thermocycler software will perform this automatically as part of
the standard analysis (see Note 15). The first derivative of the
raw data in Fig. 1a is shown in Fig. 1b.
3. Find the maximum of the first derivative, which corresponds
(approximately) to the apparent midpoint of thermal denaturation (T m ). Most thermocycler software will do this automatically, with an adjustable threshold so that noisy baseline regions
can be excluded from the search for maxima.
4. Average the T m values from replicate measurements and calculate the ΔT m according to ΔT m ¼ T m
HEWL+NAG3
À T m
HEWL
.
In five replicates of this experiment, each consisting of triplicate
samples for each condition, the mean T m for HEWL was
76.5 Æ 0.2
C and the mean T m for HEWL + 1 mM NAG3 was
79.6 Æ 0.3
C, and the mean ΔT m was 3.2 Æ 0.4
C (see Note 16).
Thus, there is clear evidence of binding of NAG3 to HEWL under
these conditions, indicated by a significant stabilization of HEWL
in the presence of 1 mM NAG3.
a
b
40
60
Temperature (°C)
Temperature (°C)
80
0
20
40
60
Fluorescence Intensity
(Arbitrary Units)
40
60
80
-5
0
5
10
First Derivative
Fig. 1 DSF raw fluorescence intensity data (a) and first derivative (b) for HEWL in the presence (red) and
absence (blue) of 1 mM NAG3
56
Xiaochun Li-Blatter et al.