9. Press “Fit.” Define the signal-to-noise ratio b
F as
b
F ¼
F n,AB
∗ À F n,B
∗
r:m:s:d:
ð6Þ
where F n,AB* and F n,B* are the refined thermophoresis
(or T-Jump) values for the AB complex and the labeled protein
alone, respectively, and r.m.s.d. is the root-mean-squared
deviation.
10. Repeat steps 6 and 7 of this section for the 40% and 60%
MST-power data (see Note 22). For future experiments,
choose the best signal-to-noise ratio; in this case (Fig. 5b), it
was for the 60% MST-power experiment (Table 1; see Note
23).
3.4 Performing
the MST Experiment
1. Fill 16 of the preferred style of capillaries with the reserved
solutions from step 2 of Subheading 3.3. Place in the capillary
tray, perform a capillary scan, and collect a single set of data
using the preferred LED power and MST power defined in
Subheadings 3.2 and 3.3, and the timing parameters delineated
in step 6 of Subheading 3.2. In this case, make sure to name
the experiment; an example is “Replicate #1.”
2. If using the NT.Control software, actuate a post-run capillary
scan. Optionally, you may save this scan. The post-run capillary
scan was conducted automatically if using MO.Control. Check
to ensure that the peak shapes have a symmetric, monomodal
appearance (see Note 13).
3. Make the same serial dilution as described in step 1 of Subheading 3.3. After a 30-min incubation in the dark, repeat
steps 1 and 2 of this section, naming the new experiment
appropriately.
4. Repeat step 3 of this section. This is the final replicate; i.e., the
experiment has now been performed in triplicate.
5. Negative control: Make the serial dilution described in step 1
of Subheading 3.3, but using 40 μM CAII instead of SBTI.
CAII is a good choice for this control because it is an unrelated
Table 1
Signal-to-noise values for the pilot experiment in Subheading 3.3 (see main text for definitions of
symbols)
MST power
F n,B*
F n,AB*
r.m.s.d.
b
F
20
953.7
956.7
0.212
14.15
40
924.0
929.3
0.324
16.35
60
894.8
902.2
0.442
16.74
MST of Protein-Ligand Interactions
173
F as
b
F ¼
F n,AB
∗ À F n,B
∗
r:m:s:d:
ð6Þ
where F n,AB* and F n,B* are the refined thermophoresis
(or T-Jump) values for the AB complex and the labeled protein
alone, respectively, and r.m.s.d. is the root-mean-squared
deviation.
10. Repeat steps 6 and 7 of this section for the 40% and 60%
MST-power data (see Note 22). For future experiments,
choose the best signal-to-noise ratio; in this case (Fig. 5b), it
was for the 60% MST-power experiment (Table 1; see Note
23).
3.4 Performing
the MST Experiment
1. Fill 16 of the preferred style of capillaries with the reserved
solutions from step 2 of Subheading 3.3. Place in the capillary
tray, perform a capillary scan, and collect a single set of data
using the preferred LED power and MST power defined in
Subheadings 3.2 and 3.3, and the timing parameters delineated
in step 6 of Subheading 3.2. In this case, make sure to name
the experiment; an example is “Replicate #1.”
2. If using the NT.Control software, actuate a post-run capillary
scan. Optionally, you may save this scan. The post-run capillary
scan was conducted automatically if using MO.Control. Check
to ensure that the peak shapes have a symmetric, monomodal
appearance (see Note 13).
3. Make the same serial dilution as described in step 1 of Subheading 3.3. After a 30-min incubation in the dark, repeat
steps 1 and 2 of this section, naming the new experiment
appropriately.
4. Repeat step 3 of this section. This is the final replicate; i.e., the
experiment has now been performed in triplicate.
5. Negative control: Make the serial dilution described in step 1
of Subheading 3.3, but using 40 μM CAII instead of SBTI.
CAII is a good choice for this control because it is an unrelated
Table 1
Signal-to-noise values for the pilot experiment in Subheading 3.3 (see main text for definitions of
symbols)
MST power
F n,B*
F n,AB*
r.m.s.d.
b
F
20
953.7
956.7
0.212
14.15
40
924.0
929.3
0.324
16.35
60
894.8
902.2
0.442
16.74
MST of Protein-Ligand Interactions
173