protein, but using a dilution factor f of 10 and ε 280 of
18,450 M
À1 cm
À1
. Make 100 μL of 40 μM SBTI, 0.05%
(v/v) Tween-20.
3. Carbonic anhydrase isoform II (CAII): Dissolve approximately
2 mg of the protein in 250 μL of PBS. As above, measure the
concentration spectrophotometrically, using a dilution factor
of 100 and ε 280 of 55,100 M
À1 cm
À1 . Make 100 μL of 40 μM
CAII, 0.05% (v/v) Tween-20.
2.3 Dye Labeling
1. 40 mM Cyanine-5 N-hydroxysuccinimide ester (Cyanine-5
NHS): Dissolve 1 mg of Cyanine-5 NHS in 37.5 μL of 100%
DMSO. Dispense the stock into 1 μL aliquots in PCR tubes
and freeze at À80
C. See Note 6.
2. A small-scale desalting gravity column, such as a PD MiniTrap
G-25 column (GE Healthcare).
2.4 Thermophoresis
Supplies
1. Standard- and Premium-coated capillaries, NanoTemper
Technologies GmbH.
2. PCR tubes.
2.5 Software/
Computer
1. PC computer equipped with Windows 7 or higher.
2. NT.Control or MO.Control Acquisition software (see Note 7).
3. PALMIST [8, 9] version 1.5.8 or higher: Available as a zipped
archive for free at http://biophysics.swmed.edu/MBR/soft
ware.html.
4. GUSSI [10] version 1.4.2 or higher: Available as a zipped
archive for free at http://biophysics.swmed.edu/MBR/soft
ware.html.
5. Complete the analysis environment by creating a folder called
C:\sedfit. Next, extract the PALMIST archive into C:\sedfit. Do
the same for the GUSSI archive. The C:\sedfit folder should
now have subfolders called “PALMIST” and “GUSSI.” The
respective executables can be found in these folders. It is convenient to make desktop shortcuts to these executable or to pin
them to the system taskbar.
2.6 Instrumentation
1. A NanoTemper NT.115 MST instrument equipped with a red
filter set.
3 Methods
Keep all protein solutions on ice unless noted. All dye-containing
solutions should be shielded from light when not in use.
MST of Protein-Ligand Interactions
165
18,450 M
À1 cm
À1
. Make 100 μL of 40 μM SBTI, 0.05%
(v/v) Tween-20.
3. Carbonic anhydrase isoform II (CAII): Dissolve approximately
2 mg of the protein in 250 μL of PBS. As above, measure the
concentration spectrophotometrically, using a dilution factor
of 100 and ε 280 of 55,100 M
À1 cm
À1 . Make 100 μL of 40 μM
CAII, 0.05% (v/v) Tween-20.
2.3 Dye Labeling
1. 40 mM Cyanine-5 N-hydroxysuccinimide ester (Cyanine-5
NHS): Dissolve 1 mg of Cyanine-5 NHS in 37.5 μL of 100%
DMSO. Dispense the stock into 1 μL aliquots in PCR tubes
and freeze at À80
C. See Note 6.
2. A small-scale desalting gravity column, such as a PD MiniTrap
G-25 column (GE Healthcare).
2.4 Thermophoresis
Supplies
1. Standard- and Premium-coated capillaries, NanoTemper
Technologies GmbH.
2. PCR tubes.
2.5 Software/
Computer
1. PC computer equipped with Windows 7 or higher.
2. NT.Control or MO.Control Acquisition software (see Note 7).
3. PALMIST [8, 9] version 1.5.8 or higher: Available as a zipped
archive for free at http://biophysics.swmed.edu/MBR/soft
ware.html.
4. GUSSI [10] version 1.4.2 or higher: Available as a zipped
archive for free at http://biophysics.swmed.edu/MBR/soft
ware.html.
5. Complete the analysis environment by creating a folder called
C:\sedfit. Next, extract the PALMIST archive into C:\sedfit. Do
the same for the GUSSI archive. The C:\sedfit folder should
now have subfolders called “PALMIST” and “GUSSI.” The
respective executables can be found in these folders. It is convenient to make desktop shortcuts to these executable or to pin
them to the system taskbar.
2.6 Instrumentation
1. A NanoTemper NT.115 MST instrument equipped with a red
filter set.
3 Methods
Keep all protein solutions on ice unless noted. All dye-containing
solutions should be shielded from light when not in use.
MST of Protein-Ligand Interactions
165