2. The effective particle theory (EPT) explains the behavior of
sedimentation boundary patterns arising in rapidly interacting
systems [25]. Zhao and colleagues [17] have showed that the
regime (slow or fast) of the association equilibrium in SV
depends on the kinetic off-rate constant (see Fig. 7 from 17).
An equilibrium with log(k off ) of À1 can be considered infinitely
fast in the context of SV. When log(k off ) ¼ À3, the equilibrium
is still fast but on the limit where kinetics should significantly
influence the boundary pattern. A value of À3.4 corresponds to
an intermediate regime, a value of À4 corresponds to the slow
regime but still influenced by kinetics, and a value of À6 means
an essentially infinitely slow regime on the SV time-scale.
3. On Day D—1 before SV experiment, GST-BTG2 was loaded
on a gel filtration column equilibrated in SV buffer to eliminate
traces of aggregates, and PABP was dialyzed overnight against
the same buffer. As TCEP is not very stable in phosphate
buffers, the SV buffer was prepared immediately before use.
Data showed that TCEP is 100% oxidized within 72 h in PBS
pH 7 and 50% oxidized within 72 h in PBS pH 8 compared to
only 20% oxidized within 3 weeks in Tris or Hepes buffers in a
broad range of pH.
4. The program SEDNTERP allows to calculate the molar mass,
partial specific volume, and extinction coefficient of proteins
from their amino acid compositions, and the solvent density
and viscosity from the solvent composition using tabulated
data. For unusual solvents, these latter parameters should be
measured experimentally using a density-meter and a viscositymeter.
5. Filling the sample compartment with a larger volume of sample
will allow to collect a larger number of scans and therefore
extract more information from the SV data. Up to 450 μL of
sample can be loaded in the cell compartment with a 12 mm
centerpiece. The volume of buffer has to match the volume of
sample in case of interference data acquisition and be equal or
higher in the case of absorbance data acquisition. There is no
need of a blank buffer sector in the case of fluorescence data
acquisition which allows both sectors of a double-sector centerpiece to be loaded with different samples.
6. Using the AN-50 Ti rotor, the maximum rotor speed is
50,000 rpm (201,600 Â g). For proteins with M < 300 kDa,
SV runs at 50,000 rpm are recommended to optimize hydrodynamic resolution. For M > 300 kDa, SV runs at lower speed
(40,000 rpm or less) are performed to maximize the number of
scans that will be collected before complete pelleting of the
macromolecule [21, 26].
168
Christine Ebel and Catherine Birck
sedimentation boundary patterns arising in rapidly interacting
systems [25]. Zhao and colleagues [17] have showed that the
regime (slow or fast) of the association equilibrium in SV
depends on the kinetic off-rate constant (see Fig. 7 from 17).
An equilibrium with log(k off ) of À1 can be considered infinitely
fast in the context of SV. When log(k off ) ¼ À3, the equilibrium
is still fast but on the limit where kinetics should significantly
influence the boundary pattern. A value of À3.4 corresponds to
an intermediate regime, a value of À4 corresponds to the slow
regime but still influenced by kinetics, and a value of À6 means
an essentially infinitely slow regime on the SV time-scale.
3. On Day D—1 before SV experiment, GST-BTG2 was loaded
on a gel filtration column equilibrated in SV buffer to eliminate
traces of aggregates, and PABP was dialyzed overnight against
the same buffer. As TCEP is not very stable in phosphate
buffers, the SV buffer was prepared immediately before use.
Data showed that TCEP is 100% oxidized within 72 h in PBS
pH 7 and 50% oxidized within 72 h in PBS pH 8 compared to
only 20% oxidized within 3 weeks in Tris or Hepes buffers in a
broad range of pH.
4. The program SEDNTERP allows to calculate the molar mass,
partial specific volume, and extinction coefficient of proteins
from their amino acid compositions, and the solvent density
and viscosity from the solvent composition using tabulated
data. For unusual solvents, these latter parameters should be
measured experimentally using a density-meter and a viscositymeter.
5. Filling the sample compartment with a larger volume of sample
will allow to collect a larger number of scans and therefore
extract more information from the SV data. Up to 450 μL of
sample can be loaded in the cell compartment with a 12 mm
centerpiece. The volume of buffer has to match the volume of
sample in case of interference data acquisition and be equal or
higher in the case of absorbance data acquisition. There is no
need of a blank buffer sector in the case of fluorescence data
acquisition which allows both sectors of a double-sector centerpiece to be loaded with different samples.
6. Using the AN-50 Ti rotor, the maximum rotor speed is
50,000 rpm (201,600 Â g). For proteins with M < 300 kDa,
SV runs at 50,000 rpm are recommended to optimize hydrodynamic resolution. For M > 300 kDa, SV runs at lower speed
(40,000 rpm or less) are performed to maximize the number of
scans that will be collected before complete pelleting of the
macromolecule [21, 26].
168
Christine Ebel and Catherine Birck