The tubes “self-fill” by capillary action. Also, fill one Standard
and one Premium capillaries with the 20 μM SBTI sample.
Arrange the eight capillaries in the instrument’s capillary tray
in the following order, starting at capillary position 1:
(a) 0 μM SBTI, Standard Capillary
(b) 0 μM SBTI, Premium Capillary
(c) 20 μM SBTI, Standard Capillary
(d) 20 μM SBTI, Premium Capillary
(e) 0 μM SBTI, Standard Capillary
(f) 0 μM SBTI, Standard Capillary
(g) 0 μM SBTI, Premium Capillary
(h) 0 μM SBTI, Premium Capillary
3. Insert the capillary tray into the instrument (see Note 12),
making sure that the red filter set is selected. If using NT.
Control, proceed to step 4. In MO.Control, perform a “Binding Test,” selecting “Auto-detect” for LED power and “Low”
MST power. The test will conduct a pre-run capillary scan (i.e.,
it will pass LED light through all capillaries sequentially), a
brief MST experiment (e.g., Fig. 1b) on all capillaries, and a
post-run capillary scan (see Note 13). It will examine the
capillary scans for overall intensity, scan shape, and intensity
trends; it examines the MST Scans for signs of aggregation, and
raises warnings if any problems occur. If a low- or highintensity warning is sounded, the LED power can be adjusted
(also see Note 14). If a scan-shape warning is raised, it likely
indicates protein sticking to the capillaries, and that defect must
be addressed before moving forward (see Note 15). An
intensity-trend warning can be ignored at this stage; however,
if there is a noticeable, systematic difference in the peak intensities between the Standard and the Premium capillaries, the
capillary type having the highest intensity should be used
henceforth. Note which LED power was selected by the software in this step. The results of the “Binding Test” should be
ignored. Repeat this step with “Medium” and “High” MST
powers, initiating a new “Binding Check” in both instances.
Proceed to step 7.
4. Using NT.Control, perform a capillary scan on the eight capillaries at 50% LED power.
5. Examine the capillary scan (Fig. 2). In our case, all of the peaks
in the scan have raw fluorescence intensity values of about 500.
This is a desirable outcome; according to the manufacturer, the
peak values should be between 200 and 2000. We usually aim
for 400–500, and the LED power can be adjusted to meet this
goal (see Note 14). Alternatively, the concentration of the
labeled protein can be modified accordingly. Other features of
MST of Protein-Ligand Interactions
167
and one Premium capillaries with the 20 μM SBTI sample.
Arrange the eight capillaries in the instrument’s capillary tray
in the following order, starting at capillary position 1:
(a) 0 μM SBTI, Standard Capillary
(b) 0 μM SBTI, Premium Capillary
(c) 20 μM SBTI, Standard Capillary
(d) 20 μM SBTI, Premium Capillary
(e) 0 μM SBTI, Standard Capillary
(f) 0 μM SBTI, Standard Capillary
(g) 0 μM SBTI, Premium Capillary
(h) 0 μM SBTI, Premium Capillary
3. Insert the capillary tray into the instrument (see Note 12),
making sure that the red filter set is selected. If using NT.
Control, proceed to step 4. In MO.Control, perform a “Binding Test,” selecting “Auto-detect” for LED power and “Low”
MST power. The test will conduct a pre-run capillary scan (i.e.,
it will pass LED light through all capillaries sequentially), a
brief MST experiment (e.g., Fig. 1b) on all capillaries, and a
post-run capillary scan (see Note 13). It will examine the
capillary scans for overall intensity, scan shape, and intensity
trends; it examines the MST Scans for signs of aggregation, and
raises warnings if any problems occur. If a low- or highintensity warning is sounded, the LED power can be adjusted
(also see Note 14). If a scan-shape warning is raised, it likely
indicates protein sticking to the capillaries, and that defect must
be addressed before moving forward (see Note 15). An
intensity-trend warning can be ignored at this stage; however,
if there is a noticeable, systematic difference in the peak intensities between the Standard and the Premium capillaries, the
capillary type having the highest intensity should be used
henceforth. Note which LED power was selected by the software in this step. The results of the “Binding Test” should be
ignored. Repeat this step with “Medium” and “High” MST
powers, initiating a new “Binding Check” in both instances.
Proceed to step 7.
4. Using NT.Control, perform a capillary scan on the eight capillaries at 50% LED power.
5. Examine the capillary scan (Fig. 2). In our case, all of the peaks
in the scan have raw fluorescence intensity values of about 500.
This is a desirable outcome; according to the manufacturer, the
peak values should be between 200 and 2000. We usually aim
for 400–500, and the LED power can be adjusted to meet this
goal (see Note 14). Alternatively, the concentration of the
labeled protein can be modified accordingly. Other features of
MST of Protein-Ligand Interactions
167