3. Balance one cell to the calibration cell and load an An-50 Ti
rotor with the cells according to Beckman Coulter rotor manual LXL/A-TB-003F provided with the centrifuge (see Note
8).
4. Install the rotor in the vacuum chamber of the centrifuge, mount
the FDS optics, and start a run at 0 rpm and 20
C at the
centrifuge (see Note 9). Switch on the AU System Box and
start the AOS (AU-Advanced Operating System) program afterward. There you have to select in the “Rotor Setup” menu the
“AnTi-8: 8-hole Titanium” rotor, the position of the calibration
cell, and “2 channel Charcoal-Epon Velocity” at the seven positions where you placed the sample containing cells.
5. When the vacuum is below 170 mTorr, the laser can be ignited
by selecting “Start” and “Start machine.” It will take about
5 min for the laser to warm up and lock in the operating state.
Successful locking of the laser can be recognized when the
“FDS Laser” signal of the “Optical System Status” box turns
from cyan to green.
6. Accelerate the rotor to 3000 rpm. After the “Magnet Angle”
display turned green, use “Fluorescence focusing” from the
“Fluorescence” menu to measure a focus scan of the calibration
strip and perform a radial scan at the optimal focus position.
Check whether the outer edge of the calibration strip is at the
expected position. Otherwise, save the radial calibration, repeat
the radial scan, and check the position of the outer edge again;
it should now be correct. Perform a focus scan of one of the
sample containing channels and save the focus (see Note 10).
7. Choose “Set gains” from the “Fluorescence” menu and display
the signal of all cells. Now adjust the voltage of the photomultiplier (0–100%) and the gain (1–8) in order to get the best
signal to noise ratio. The signal can be in the range of 0–4000,
which is the upper limit of the photomultiplier. Since the
fluorophore concentration in all samples is the same, there
should be no need to use different gain settings (see Note 11).
8. Perform one scan at 3000 rpm in order to check whether your
cells are properly filled and whether all settings are correct. Use
the “Stop” button in AOS and select “Stop the machine” to
switch off the laser and to stop the rotor. Since this will also
turn off the diffusion pump of the centrifuge, instantly start a
run at 0 rpm at the centrifuge (see Note 9) and wait for thermal
equilibration at least for 1 h after the temperature of 20
C is
displayed at the machine.
9. Switch on the laser by using the “Start” button of AOS and
select “Start machine.” Create a new method using “Select
method” with a rotor speed of 40,000 rpm (see Note 12),
10 h duration, and 20
C and set “interval between scans” to
Analysis of Protein-DNA Interactions by AUC
413
rotor with the cells according to Beckman Coulter rotor manual LXL/A-TB-003F provided with the centrifuge (see Note
8).
4. Install the rotor in the vacuum chamber of the centrifuge, mount
the FDS optics, and start a run at 0 rpm and 20
C at the
centrifuge (see Note 9). Switch on the AU System Box and
start the AOS (AU-Advanced Operating System) program afterward. There you have to select in the “Rotor Setup” menu the
“AnTi-8: 8-hole Titanium” rotor, the position of the calibration
cell, and “2 channel Charcoal-Epon Velocity” at the seven positions where you placed the sample containing cells.
5. When the vacuum is below 170 mTorr, the laser can be ignited
by selecting “Start” and “Start machine.” It will take about
5 min for the laser to warm up and lock in the operating state.
Successful locking of the laser can be recognized when the
“FDS Laser” signal of the “Optical System Status” box turns
from cyan to green.
6. Accelerate the rotor to 3000 rpm. After the “Magnet Angle”
display turned green, use “Fluorescence focusing” from the
“Fluorescence” menu to measure a focus scan of the calibration
strip and perform a radial scan at the optimal focus position.
Check whether the outer edge of the calibration strip is at the
expected position. Otherwise, save the radial calibration, repeat
the radial scan, and check the position of the outer edge again;
it should now be correct. Perform a focus scan of one of the
sample containing channels and save the focus (see Note 10).
7. Choose “Set gains” from the “Fluorescence” menu and display
the signal of all cells. Now adjust the voltage of the photomultiplier (0–100%) and the gain (1–8) in order to get the best
signal to noise ratio. The signal can be in the range of 0–4000,
which is the upper limit of the photomultiplier. Since the
fluorophore concentration in all samples is the same, there
should be no need to use different gain settings (see Note 11).
8. Perform one scan at 3000 rpm in order to check whether your
cells are properly filled and whether all settings are correct. Use
the “Stop” button in AOS and select “Stop the machine” to
switch off the laser and to stop the rotor. Since this will also
turn off the diffusion pump of the centrifuge, instantly start a
run at 0 rpm at the centrifuge (see Note 9) and wait for thermal
equilibration at least for 1 h after the temperature of 20
C is
displayed at the machine.
9. Switch on the laser by using the “Start” button of AOS and
select “Start machine.” Create a new method using “Select
method” with a rotor speed of 40,000 rpm (see Note 12),
10 h duration, and 20
C and set “interval between scans” to
Analysis of Protein-DNA Interactions by AUC
413