8. For MST experiments, it is recommended to use low binding
tubes and tips. For the serial dilution, better results are
obtained without changing the tips from tube to tube. However, when adding the labeling molecule, it is recommended to
change the tip at each tube. Avoid making bubbles by pipetting
carefully and never vortex these small volumes to avoid denaturation of samples. Handle capillaries with care and never
touch at the center where the measurement is performed.
9. An SDS denaturation test (SD-test) has to be performed to
distinguish between fluorescence changes caused by an interaction and those caused by nonspecific effects, e.g., loss of protein due to aggregation or adsorption to labware. This test
consists in the denaturation of all proteins contained in the
sample using a mix of SDS and DTT and heating to 95
C. In
this context, the receptor–ligand interaction is disrupted. After
denaturation, the change of fluorescence should not be
observed in the absence or in the presence of increasing concentration of ligands. If addition of ligands induces fluorescence change with denatured protein, this suggests some
interference of the ligand and some new experimental conditions should be researched.
10. If not enough fluorescence is observed, one can increase the
concentration of the labeled receptor or increase the LED
power. A different strategy of labeling can be used. In cases of
absorption to the capillary, adding detergents or other amphiphilic polymer additives will help.
11. Under certain conditions, MST can also be used to obtain the
stoichiometry of the interaction. For that, we need to know
precisely the concentration of receptor and ligand as well as the
K d of their interaction. In this case, we need a much higher
concentration of the receptor at least 20-fold than the K d .
12. Thermodynamic can be extracted thanks to the Van’t Hoff
equation: ln K D ¼
ΔH
ÀRT þ
ΔS
R . When plotting ln(K D ) ¼ f
(1/T), one can obtain a straight line which can be fitted as a
linear equation where the slope is given by ΔH/ÀR and the
intercept is ΔS/R.
13. It can be worth to label the other molecule and repeat the
experiment in the reverse way to confirm the result.
14. A positive control and a negative control are advisable to
confirm that there is no unspecific interaction or that the
conditions of the assay are optimal.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
141
tubes and tips. For the serial dilution, better results are
obtained without changing the tips from tube to tube. However, when adding the labeling molecule, it is recommended to
change the tip at each tube. Avoid making bubbles by pipetting
carefully and never vortex these small volumes to avoid denaturation of samples. Handle capillaries with care and never
touch at the center where the measurement is performed.
9. An SDS denaturation test (SD-test) has to be performed to
distinguish between fluorescence changes caused by an interaction and those caused by nonspecific effects, e.g., loss of protein due to aggregation or adsorption to labware. This test
consists in the denaturation of all proteins contained in the
sample using a mix of SDS and DTT and heating to 95
C. In
this context, the receptor–ligand interaction is disrupted. After
denaturation, the change of fluorescence should not be
observed in the absence or in the presence of increasing concentration of ligands. If addition of ligands induces fluorescence change with denatured protein, this suggests some
interference of the ligand and some new experimental conditions should be researched.
10. If not enough fluorescence is observed, one can increase the
concentration of the labeled receptor or increase the LED
power. A different strategy of labeling can be used. In cases of
absorption to the capillary, adding detergents or other amphiphilic polymer additives will help.
11. Under certain conditions, MST can also be used to obtain the
stoichiometry of the interaction. For that, we need to know
precisely the concentration of receptor and ligand as well as the
K d of their interaction. In this case, we need a much higher
concentration of the receptor at least 20-fold than the K d .
12. Thermodynamic can be extracted thanks to the Van’t Hoff
equation: ln K D ¼
ΔH
ÀRT þ
ΔS
R . When plotting ln(K D ) ¼ f
(1/T), one can obtain a straight line which can be fitted as a
linear equation where the slope is given by ΔH/ÀR and the
intercept is ΔS/R.
13. It can be worth to label the other molecule and repeat the
experiment in the reverse way to confirm the result.
14. A positive control and a negative control are advisable to
confirm that there is no unspecific interaction or that the
conditions of the assay are optimal.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
141