in fluorescence intensity with increasing focus depth is
observed, this is a sign of an inner-filter effect (see Subheading
1.1), indicating that the fluorophore concentration is too high
or that the sample scatters light significantly.
11. If the samples differ significantly in fluorescence intensity, different settings can be used for different cells. Since a change of
the PMT voltage will make it impossible to compare the signals
from different cells, it is recommended to change the gains
only. However, it has to be taken into account that each extra
setting requires an extra scan round for those cells, thereby
limiting the number of scans that can be taken from an individual cell before the sample is completely sedimented.
12. For AUC, centrifuge settings are quoted in terms of rpm rather
than g, as the g-force on a sedimenting particle depends on its
radial position, and the concentration of the particle is
measured as a function of the distance from the center of
rotation. Therefore, no single value for the g-force can be
given. A rotor speed of 40,000 rpm is optimal for analysis of
the interaction of the clamp loader and the SSB-saturated
template/primer DNAs we used in this analysis. If you use
other proteins or DNAs, it might be necessary to adjust the
rotor speed to higher or lower values. The maximal speed of
the An-50 Ti rotor is 50,000 rpm. Be aware that sedimentation
velocity increases quadratically with rotor speed.
13. We found that the stability of the signal is improved if the laser
is turned on 30 min before the start of the experiment. Make
sure not to wait any longer since after the system is idle for
30 min, the laser will turn off automatically.
14. In AUC with fluorescence detection, the meniscus position is
not as easy to see as in absorbance measurements where due to
light diffraction a noticeable optical artifact can be seen. While
usually there is no problem to determine the exact meniscus
position by fitting in SEDFIT, it is sometimes helpful to measure the meniscus position by an absorbance scan after the
actual run. In such cases, it is best to mount the absorbance
optics together with the fluorescence optics before the run and
to switch after the run, without stopping the rotor, to the
Beckman Coulter control software and take absorbance scans
from all cells at 320 nm. Since both sectors are filled with
samples, intensity mode should be used.
15. If the reaction is slow on the time scale of sedimentation, no
reaction boundary will be observed and both interaction partners and the complex will sediment in extra boundaries. Since
only one reaction partner is labeled, in this case two peaks are
expected in the c(s) distributions: one sedimenting with the
s-value of the free labeled macromolecule and the other one
418
Andrea Bogutzki and Ute Curth
observed, this is a sign of an inner-filter effect (see Subheading
1.1), indicating that the fluorophore concentration is too high
or that the sample scatters light significantly.
11. If the samples differ significantly in fluorescence intensity, different settings can be used for different cells. Since a change of
the PMT voltage will make it impossible to compare the signals
from different cells, it is recommended to change the gains
only. However, it has to be taken into account that each extra
setting requires an extra scan round for those cells, thereby
limiting the number of scans that can be taken from an individual cell before the sample is completely sedimented.
12. For AUC, centrifuge settings are quoted in terms of rpm rather
than g, as the g-force on a sedimenting particle depends on its
radial position, and the concentration of the particle is
measured as a function of the distance from the center of
rotation. Therefore, no single value for the g-force can be
given. A rotor speed of 40,000 rpm is optimal for analysis of
the interaction of the clamp loader and the SSB-saturated
template/primer DNAs we used in this analysis. If you use
other proteins or DNAs, it might be necessary to adjust the
rotor speed to higher or lower values. The maximal speed of
the An-50 Ti rotor is 50,000 rpm. Be aware that sedimentation
velocity increases quadratically with rotor speed.
13. We found that the stability of the signal is improved if the laser
is turned on 30 min before the start of the experiment. Make
sure not to wait any longer since after the system is idle for
30 min, the laser will turn off automatically.
14. In AUC with fluorescence detection, the meniscus position is
not as easy to see as in absorbance measurements where due to
light diffraction a noticeable optical artifact can be seen. While
usually there is no problem to determine the exact meniscus
position by fitting in SEDFIT, it is sometimes helpful to measure the meniscus position by an absorbance scan after the
actual run. In such cases, it is best to mount the absorbance
optics together with the fluorescence optics before the run and
to switch after the run, without stopping the rotor, to the
Beckman Coulter control software and take absorbance scans
from all cells at 320 nm. Since both sectors are filled with
samples, intensity mode should be used.
15. If the reaction is slow on the time scale of sedimentation, no
reaction boundary will be observed and both interaction partners and the complex will sediment in extra boundaries. Since
only one reaction partner is labeled, in this case two peaks are
expected in the c(s) distributions: one sedimenting with the
s-value of the free labeled macromolecule and the other one
418
Andrea Bogutzki and Ute Curth