and MP/surfactant complexes are easily discriminated, even for relatively small particles whose hydrodynamic radii (R S ) are not necessarily very different from one another. For example, the sedimentation
coefficients of A8-35 particles (R S % 3.15 nm; Gohon et al. 2006) and BR/A8-35 complexes (R S % 5.0 nm;
Gohon et al. 2008) are 1.6 S and 3.2 S, respectively, making them easily distinguishable. The higher
sensitivity of SV-AUC is the consequence of s being proportional to the ratio of the buoyant molar mass
over R S , which is roughly proportional to M
2/3
, with M the molar mass, whereas size exclusion
chromatography (SEC) separates particles according to R S , which varies roughly as M
1/3
. As regards
determining the mass of APol bound per particle, AUC is technologically more demanding than SEC or
IMAC, but it presents the advantage that MP/APol complexes are never separated from the free APol,
which sediments more slowly, thus eliminating the risks of desorption and/or aggregation.
9.5.1.2 Instrumentation
AUC experiments require access to an analytical ultracentrifuge (XLI Beckman, USA), a rotor (AnTi
50 or 60), and cell assemblies with sapphire windows (e.g. from Nanolytik, Germany).
9.5.1.3 Designing the Samples and Experiments
There is some flexibility in terms of protein and APol concentrations. Typically, samples are examined
at several different concentrations in order to look for the presence of eventual association/dissociation
processes and to check on the results. A sample containing only APol can be included so as to probe its
behavior.
Available cell centerpieces have optical path lengths of 1.2, 0.3, or 0.15 mm, requiring volumes of
450, 150, and 80 μL, respectively. The sedimentation can be followed at a maximum of three
wavelengths in the range 230–600 nm, preferably at maxima in the absorbance spectrum. Typically,
sedimentation profiles are recorded by measuring the absorbance at 280 nm and at a second
wavelength if relevant (e.g. if the protein contains a chromophore or binds a partner absorbing at a
different wavelength, or if one resorts to a labeled APol), as well as by interference optics.
The absorbance of the stock sample has to be measured and an aliquot diluted to achieve a final
absorbance in the centrifuge between 1.2 and 0.2 (it can be raised to 1.4 depending on AUC optics
quality or lowered to 0.05 if the sample is highly homogeneous). Usually, we dilute the stock
sample, which typically is in the g‧L
À1 range, by a factor of two to four. These concentrations are
also convenient for interference measurements. In principle, for maintaining the chemical potential
of APol constant in the dilution series, the buffer used for dilution should contain free APol at the
same concentration as in the stock sample, but this is not necessarily an issue. Note that above
~2 g‧L
À1 , samples behave non-ideally, which will have to be taken into account in the analysis of
results obtained at different dilutions (Solovyova et al. 2001).
The buffer should not absorb, or only slightly so, at the wavelengths of interest, and should
contain typically 100 mM salt to avoid repulsive electrostatic interactions. AUC centerpieces have
two channels, one of which is to be filled with the reference buffer. The reference buffer should have
exactly the same composition as the sample buffer (it is particularly important for buffers
containing large amount of, for instance, glycerol), but for the omission of APol.
9.5 Protocols
441
coefficients of A8-35 particles (R S % 3.15 nm; Gohon et al. 2006) and BR/A8-35 complexes (R S % 5.0 nm;
Gohon et al. 2008) are 1.6 S and 3.2 S, respectively, making them easily distinguishable. The higher
sensitivity of SV-AUC is the consequence of s being proportional to the ratio of the buoyant molar mass
over R S , which is roughly proportional to M
2/3
, with M the molar mass, whereas size exclusion
chromatography (SEC) separates particles according to R S , which varies roughly as M
1/3
. As regards
determining the mass of APol bound per particle, AUC is technologically more demanding than SEC or
IMAC, but it presents the advantage that MP/APol complexes are never separated from the free APol,
which sediments more slowly, thus eliminating the risks of desorption and/or aggregation.
9.5.1.2 Instrumentation
AUC experiments require access to an analytical ultracentrifuge (XLI Beckman, USA), a rotor (AnTi
50 or 60), and cell assemblies with sapphire windows (e.g. from Nanolytik, Germany).
9.5.1.3 Designing the Samples and Experiments
There is some flexibility in terms of protein and APol concentrations. Typically, samples are examined
at several different concentrations in order to look for the presence of eventual association/dissociation
processes and to check on the results. A sample containing only APol can be included so as to probe its
behavior.
Available cell centerpieces have optical path lengths of 1.2, 0.3, or 0.15 mm, requiring volumes of
450, 150, and 80 μL, respectively. The sedimentation can be followed at a maximum of three
wavelengths in the range 230–600 nm, preferably at maxima in the absorbance spectrum. Typically,
sedimentation profiles are recorded by measuring the absorbance at 280 nm and at a second
wavelength if relevant (e.g. if the protein contains a chromophore or binds a partner absorbing at a
different wavelength, or if one resorts to a labeled APol), as well as by interference optics.
The absorbance of the stock sample has to be measured and an aliquot diluted to achieve a final
absorbance in the centrifuge between 1.2 and 0.2 (it can be raised to 1.4 depending on AUC optics
quality or lowered to 0.05 if the sample is highly homogeneous). Usually, we dilute the stock
sample, which typically is in the g‧L
À1 range, by a factor of two to four. These concentrations are
also convenient for interference measurements. In principle, for maintaining the chemical potential
of APol constant in the dilution series, the buffer used for dilution should contain free APol at the
same concentration as in the stock sample, but this is not necessarily an issue. Note that above
~2 g‧L
À1 , samples behave non-ideally, which will have to be taken into account in the analysis of
results obtained at different dilutions (Solovyova et al. 2001).
The buffer should not absorb, or only slightly so, at the wavelengths of interest, and should
contain typically 100 mM salt to avoid repulsive electrostatic interactions. AUC centerpieces have
two channels, one of which is to be filled with the reference buffer. The reference buffer should have
exactly the same composition as the sample buffer (it is particularly important for buffers
containing large amount of, for instance, glycerol), but for the omission of APol.
9.5 Protocols
441