110 μL of buffer (see Note 5). Close the cells and check that
cells that will be placed in opposite positions in the rotor have
the same weights (tolerance of 0.5 g). Place and align the cells
in the rotor holes.
2. Prepare the instrument: Put the rotor then the optical arm into
the analytical ultracentrifuge, and initiate vacuum and temperature equilibration. Wait at least 2 h at the requested temperature before starting the SV experiment.
3. Check the absorbance signal in each cell. During temperature
equilibration, start the program ProteomeLab XL-I. Give a
title for each cell and a common folder for the collected data.
Start the AUC at 3000 rpm and acquire for each cell a radial
scan at 280 nm to check that the absorbance signal corresponds
to expectation.
4. Start the SV run: In the method window, enter 150 for the
number of scans. In the options window, enter 3 for the last
scans overlay and select stop XL after the last scan. Start the
centrifuge at the chosen speed (see Note 6) then start the
method scan. After the stop of the ultracentrifuge, cells are
disassembled, cleaned, and reassembled. Raw data are copied
for analysis. A detailed protocol can be found in [8].
3.2 Non-interacting
System Data Analysis
with BSA Data Sets
We perform heterogeneity analysis of two BSA samples using both c
(s) and non-interacting species models to illustrate the potential
and limits of these approaches. The first sample, BSA1, which
contains aging aggregates is heterogeneous while the second,
BSA2, freshly prepared from size exclusion chromatography and
composed of monomeric BSA, is homogenous.
1. The c(s) analysis: Open the program SEDFIT and load a set of
SV scans corresponding to the whole sedimentation process
(Fig. 1). Set the meniscus with the red line (air-sample interface) and the radial limits for the fit with the green lines.
Choose the continuous c(s) distribution model. In the parameter window, enter the experimental values for partial specific
volume, density, and viscosity, change the confidence level to
0.68 (see Note 7) and mark the frictional ratio and meniscus
check boxes. Use the default values for all other parameters
(100 for resolution, 0–20 S for sedimentation coefficient
range, 1.2 for frictional ratio value) and press the run command
to perform a simulation of the sedimentation process using the
given parameters. The simulated data are displayed as lines with
the loaded scans, and a first c(s) distribution plot appears. Then
press the fit command to optimize the checked parameters
(frictional ratio and meniscus position). After convergence,
simulated data should fit very well to the experimental data
162
Christine Ebel and Catherine Birck
cells that will be placed in opposite positions in the rotor have
the same weights (tolerance of 0.5 g). Place and align the cells
in the rotor holes.
2. Prepare the instrument: Put the rotor then the optical arm into
the analytical ultracentrifuge, and initiate vacuum and temperature equilibration. Wait at least 2 h at the requested temperature before starting the SV experiment.
3. Check the absorbance signal in each cell. During temperature
equilibration, start the program ProteomeLab XL-I. Give a
title for each cell and a common folder for the collected data.
Start the AUC at 3000 rpm and acquire for each cell a radial
scan at 280 nm to check that the absorbance signal corresponds
to expectation.
4. Start the SV run: In the method window, enter 150 for the
number of scans. In the options window, enter 3 for the last
scans overlay and select stop XL after the last scan. Start the
centrifuge at the chosen speed (see Note 6) then start the
method scan. After the stop of the ultracentrifuge, cells are
disassembled, cleaned, and reassembled. Raw data are copied
for analysis. A detailed protocol can be found in [8].
3.2 Non-interacting
System Data Analysis
with BSA Data Sets
We perform heterogeneity analysis of two BSA samples using both c
(s) and non-interacting species models to illustrate the potential
and limits of these approaches. The first sample, BSA1, which
contains aging aggregates is heterogeneous while the second,
BSA2, freshly prepared from size exclusion chromatography and
composed of monomeric BSA, is homogenous.
1. The c(s) analysis: Open the program SEDFIT and load a set of
SV scans corresponding to the whole sedimentation process
(Fig. 1). Set the meniscus with the red line (air-sample interface) and the radial limits for the fit with the green lines.
Choose the continuous c(s) distribution model. In the parameter window, enter the experimental values for partial specific
volume, density, and viscosity, change the confidence level to
0.68 (see Note 7) and mark the frictional ratio and meniscus
check boxes. Use the default values for all other parameters
(100 for resolution, 0–20 S for sedimentation coefficient
range, 1.2 for frictional ratio value) and press the run command
to perform a simulation of the sedimentation process using the
given parameters. The simulated data are displayed as lines with
the loaded scans, and a first c(s) distribution plot appears. Then
press the fit command to optimize the checked parameters
(frictional ratio and meniscus position). After convergence,
simulated data should fit very well to the experimental data
162
Christine Ebel and Catherine Birck