“Experimental Parameters” window will pop up and you have
to tick “EPT sw fast” and set the extinction coefficient of A to
zero, while choosing an arbitrary extinction coefficient for B
(e.g., 1), the only molecule that contributes to the fluorescence
signal.
6. Save the *.xp file as suggested and choose from the “Model”
menu “A + B $ AB Hetero-Association.”
7. Open the “Global Parameters” window and give the correct
value for the sedimentation coefficient of B (see Note 18) and
estimated values for s AB and log K A (see Note 19). Tick the
boxes of s AB and log K A to tell the program that these are the
values that should be fitted.
8. Choose “Single Experiment Run” from the “Run” menu to
check whether your settings are reasonable. If they are not,
change the estimated values, repeat this step, and if the results
are satisfying, choose “Single Experiment Fit” from the “Fit”
menu. The results of your fit will be displayed (see Note 20).
Check the quality of the fit and look whether the residuals are
evenly distributed. The highest s-value measured for the reaction boundary should be close to s AB ; if this is not the case,
not the whole isotherm is covered and it would be advisable to
repeat the experiment at higher concentrations of A. The
results of fitting binding isotherms to our data obtained for
the interaction of 100 nM FITC-CL with t/p 55/30-SSB, t/p
80/30-SSB, and SSB alone can be seen in Fig. 5b.
4 Notes
1. Especially when working with fluorescently labeled macromolecules at low nanomolar concentrations, the addition of an
inert carrier protein to your sample is recommended to avoid
sticking of labeled protein to surfaces of, e.g., the AUC cell
assembly, pipette tips, and reaction tubes. We used BSA at a
concentration of 1 μM (0.066 mg/mL). Alternatively, κ-casein
or lysozyme at concentrations of 0.1 mg/mL and 0.2 mg/mL
[32], respectively, or low concentrations of nonionic detergents like 0.05% (v/v) Tween 20 can be used.
2. In the AU-FDS calibration cell, the rectangular channel that is
positioned in the middle of the centerpiece is named channel A
(the so-called calibration strip). Channel A is used for angular
location of each cell channel, and the bottom edge of this
channel is used for radial calibration. The detection of the
signal from the calibration cell is crucial to any measurement
with the FDS. Therefore, it is very important to fill channel A
with a fluorescing solution that produces a stable signal that can
Analysis of Protein-DNA Interactions by AUC
415
to tick “EPT sw fast” and set the extinction coefficient of A to
zero, while choosing an arbitrary extinction coefficient for B
(e.g., 1), the only molecule that contributes to the fluorescence
signal.
6. Save the *.xp file as suggested and choose from the “Model”
menu “A + B $ AB Hetero-Association.”
7. Open the “Global Parameters” window and give the correct
value for the sedimentation coefficient of B (see Note 18) and
estimated values for s AB and log K A (see Note 19). Tick the
boxes of s AB and log K A to tell the program that these are the
values that should be fitted.
8. Choose “Single Experiment Run” from the “Run” menu to
check whether your settings are reasonable. If they are not,
change the estimated values, repeat this step, and if the results
are satisfying, choose “Single Experiment Fit” from the “Fit”
menu. The results of your fit will be displayed (see Note 20).
Check the quality of the fit and look whether the residuals are
evenly distributed. The highest s-value measured for the reaction boundary should be close to s AB ; if this is not the case,
not the whole isotherm is covered and it would be advisable to
repeat the experiment at higher concentrations of A. The
results of fitting binding isotherms to our data obtained for
the interaction of 100 nM FITC-CL with t/p 55/30-SSB, t/p
80/30-SSB, and SSB alone can be seen in Fig. 5b.
4 Notes
1. Especially when working with fluorescently labeled macromolecules at low nanomolar concentrations, the addition of an
inert carrier protein to your sample is recommended to avoid
sticking of labeled protein to surfaces of, e.g., the AUC cell
assembly, pipette tips, and reaction tubes. We used BSA at a
concentration of 1 μM (0.066 mg/mL). Alternatively, κ-casein
or lysozyme at concentrations of 0.1 mg/mL and 0.2 mg/mL
[32], respectively, or low concentrations of nonionic detergents like 0.05% (v/v) Tween 20 can be used.
2. In the AU-FDS calibration cell, the rectangular channel that is
positioned in the middle of the centerpiece is named channel A
(the so-called calibration strip). Channel A is used for angular
location of each cell channel, and the bottom edge of this
channel is used for radial calibration. The detection of the
signal from the calibration cell is crucial to any measurement
with the FDS. Therefore, it is very important to fill channel A
with a fluorescing solution that produces a stable signal that can
Analysis of Protein-DNA Interactions by AUC
415