can still be applied through the filling holes. However, lowering the focus height of the FDS to that position would worsen
the shadowing effect by the centerpiece and the screw ring [34]
and would interfere with the detection of the signal of the
calibration strip. Therefore, use of special fluorescence cell
housings available from Nanolytics Instruments, where the
filling holes are shifted by 4.5 mm to the top of the housing
and a 9 mm elevation is included at the bottom of the housing
(Fig. 1c), is recommended. These allow the use of 3-mm
centerpieces with the FDS without the need for shifting the
focus height and for any extra spacers. Alternatively, fluorescence housings can be purchased from SpinAnalytical. However, in this case, the use of additional spacers is necessary since
the bottom of the housing is not adjusted.
6. When looking on the screw ring of an AUC cell with the screw
holes facing the top, the AOS software of the FDS will name
the left channel A whereas the right channel will be named B.
7. It may be worth reserving one channel in the run for a buffer
reference to check whether there are fluorescent sedimenting
impurities in the reaction buffer.
8. There is no need to place the FDS calibration cell in a special
hole of the rotor since the position of the calibration cell can be
freely chosen in the “Rotor Setup” menu. However, if you
would like to measure the position of the menisci after the
run with the absorbance optics of the centrifuge, you should
avoid placing the calibration cell in hole 8 since this may
interfere with delay calibration. In case you want to use both
optics, you have to mount the absorbance optics before the
FDS optics.
9. If you just press the “vacuum” button for starting evacuation
and temperature equilibration instead of pressing the “start”
button, only the vacuum pump, but not the diffusion pump, is
turned on. The actual rotor temperature, however, can only be
measured if the vacuum is below 100 mTorr; therefore, temperature equilibration might be delayed. Above 100 mTorr, the
temperature of the vacuum chamber is measured that might
substantially deviate from the rotor temperature.
10. Since the calibration strip is quite shallow and the z-resolution
of the FDS is low, the intensity of its focus scan is bell-shaped
and the maximum represents the optimal focus position. This
position is above the optimal focus position of a sample containing centerpiece. Therefore, it is recommended to perform
the actual measurements at the optimal focus position of the
sample containing cells. If 3 mm centerpieces are used, a bellshaped curve is also expected due to the low z-resolution of the
FDS. In case when 12 mm centerpieces are used and a decrease
Analysis of Protein-DNA Interactions by AUC
417
the shadowing effect by the centerpiece and the screw ring [34]
and would interfere with the detection of the signal of the
calibration strip. Therefore, use of special fluorescence cell
housings available from Nanolytics Instruments, where the
filling holes are shifted by 4.5 mm to the top of the housing
and a 9 mm elevation is included at the bottom of the housing
(Fig. 1c), is recommended. These allow the use of 3-mm
centerpieces with the FDS without the need for shifting the
focus height and for any extra spacers. Alternatively, fluorescence housings can be purchased from SpinAnalytical. However, in this case, the use of additional spacers is necessary since
the bottom of the housing is not adjusted.
6. When looking on the screw ring of an AUC cell with the screw
holes facing the top, the AOS software of the FDS will name
the left channel A whereas the right channel will be named B.
7. It may be worth reserving one channel in the run for a buffer
reference to check whether there are fluorescent sedimenting
impurities in the reaction buffer.
8. There is no need to place the FDS calibration cell in a special
hole of the rotor since the position of the calibration cell can be
freely chosen in the “Rotor Setup” menu. However, if you
would like to measure the position of the menisci after the
run with the absorbance optics of the centrifuge, you should
avoid placing the calibration cell in hole 8 since this may
interfere with delay calibration. In case you want to use both
optics, you have to mount the absorbance optics before the
FDS optics.
9. If you just press the “vacuum” button for starting evacuation
and temperature equilibration instead of pressing the “start”
button, only the vacuum pump, but not the diffusion pump, is
turned on. The actual rotor temperature, however, can only be
measured if the vacuum is below 100 mTorr; therefore, temperature equilibration might be delayed. Above 100 mTorr, the
temperature of the vacuum chamber is measured that might
substantially deviate from the rotor temperature.
10. Since the calibration strip is quite shallow and the z-resolution
of the FDS is low, the intensity of its focus scan is bell-shaped
and the maximum represents the optimal focus position. This
position is above the optimal focus position of a sample containing centerpiece. Therefore, it is recommended to perform
the actual measurements at the optimal focus position of the
sample containing cells. If 3 mm centerpieces are used, a bellshaped curve is also expected due to the low z-resolution of the
FDS. In case when 12 mm centerpieces are used and a decrease
Analysis of Protein-DNA Interactions by AUC
417