the presence of APol in equilibration and elution buffers at 5–10% that in the sample may be
advisable, so as to prevent APol desorption.
• Desalt the sample to remove imidazole and measure the optical density of the sample at
280 nm and at the maximum absorbance wavelength of FAPol. The concentrations of MP and
FAPol are calculated using their respective extinction coefficients. Subtract, if need be, the
contribution of FAPol at 280 nm, and calculate the total mass of APol if FAPol was initially
mixed with A8-35 before trapping. The ratio of APol and MP masses gives the amount of
bound APol per MP.
Method 3. Analytical Ultracentrifugation (AUC)
The MP/APol mass ratio in complexes can be precisely determined by sedimentation velocity
(SV) measurements using AUC. AUC is a priori applicable to any MP, because the density of A8-35
particles and that of MP/APol complexes are different enough for them to separate during the centrifugation run, even if their hydrodynamic radii are not very different (see Chap. 9, Protocol 9.1). For
example, the sedimentation coefficients (s) 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 specific volume of the sodium salt of A8-35,
v 2 , is 0.809 LÁg
À1
, its density,
ρ ¼ 1/
v 2 , 1.236 LÁg
À1 (Gohon et al. 2004, 2006). The MP/APol mass ratio can be determined by
sophisticated AUC measurements involving the comparison of sedimentation properties of complexes
formed between the protein and unlabeled or deuterated A8-35 and/or simultaneous measurements of
the absorbance and refractive index of the complexes (Gohon et al. 2008) (see Chap. 9). However, with
the advent of FAPols, it is simpler to measure the respective absorbance of the protein and the APol in
the complexes, as done above for the complexes separated by SEC or affinity chromatography.
• After MP trapping in the FAPol/A8-35 mixture, adjust the sample concentration by dilution
or concentration so that the protein absorbance at 280 nm, in the AUC cell, reaches ~0.5.
• Define the parameters of the SV run, namely time and speed, according to the sedimentation
coefficient of the protein under study. For instance, in the case of small MPs, like BR or tOmpA,
the SV experiment is carried out at 42,000 rpm during 4 h. The migration of the particles and
complexes is followed at two wavelengths, 280 nm and the maximum absorbance wavelength of
FAPol, and, if available, with interference optics, which give a measure of the refractive index.
• Measure the solvent density and viscosity.
• Analyze the SV profiles with Sedfit or an equivalent program (for details, see Gohon et al.
2008, and Chap. 9, Protocol 9.1). The distribution c(s) of sedimentation coefficients (s) shows
peaks reflecting the migration of MP/APol complexes and of free APol particles during the
SV run. Integrate the peak areas at 280 nm and at the second wavelength. As noted above for
SEC and IMAC experiments, the contribution of APols at 280 nm may have to be subtracted.
The ratio of MP and APol in the complexes can be determined from their respective extinction
coefficients.
Protocol prepared by Manuela Zoonens on the basis of Zoonens et al. (2007), Gohon et al.
(2008), Charvolin et al. (2014), and laboratory notes.
318
5 Formation and Properties of Membrane Protein/Amphipol Complexes
advisable, so as to prevent APol desorption.
• Desalt the sample to remove imidazole and measure the optical density of the sample at
280 nm and at the maximum absorbance wavelength of FAPol. The concentrations of MP and
FAPol are calculated using their respective extinction coefficients. Subtract, if need be, the
contribution of FAPol at 280 nm, and calculate the total mass of APol if FAPol was initially
mixed with A8-35 before trapping. The ratio of APol and MP masses gives the amount of
bound APol per MP.
Method 3. Analytical Ultracentrifugation (AUC)
The MP/APol mass ratio in complexes can be precisely determined by sedimentation velocity
(SV) measurements using AUC. AUC is a priori applicable to any MP, because the density of A8-35
particles and that of MP/APol complexes are different enough for them to separate during the centrifugation run, even if their hydrodynamic radii are not very different (see Chap. 9, Protocol 9.1). For
example, the sedimentation coefficients (s) 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 specific volume of the sodium salt of A8-35,
v 2 , is 0.809 LÁg
À1
, its density,
ρ ¼ 1/
v 2 , 1.236 LÁg
À1 (Gohon et al. 2004, 2006). The MP/APol mass ratio can be determined by
sophisticated AUC measurements involving the comparison of sedimentation properties of complexes
formed between the protein and unlabeled or deuterated A8-35 and/or simultaneous measurements of
the absorbance and refractive index of the complexes (Gohon et al. 2008) (see Chap. 9). However, with
the advent of FAPols, it is simpler to measure the respective absorbance of the protein and the APol in
the complexes, as done above for the complexes separated by SEC or affinity chromatography.
• After MP trapping in the FAPol/A8-35 mixture, adjust the sample concentration by dilution
or concentration so that the protein absorbance at 280 nm, in the AUC cell, reaches ~0.5.
• Define the parameters of the SV run, namely time and speed, according to the sedimentation
coefficient of the protein under study. For instance, in the case of small MPs, like BR or tOmpA,
the SV experiment is carried out at 42,000 rpm during 4 h. The migration of the particles and
complexes is followed at two wavelengths, 280 nm and the maximum absorbance wavelength of
FAPol, and, if available, with interference optics, which give a measure of the refractive index.
• Measure the solvent density and viscosity.
• Analyze the SV profiles with Sedfit or an equivalent program (for details, see Gohon et al.
2008, and Chap. 9, Protocol 9.1). The distribution c(s) of sedimentation coefficients (s) shows
peaks reflecting the migration of MP/APol complexes and of free APol particles during the
SV run. Integrate the peak areas at 280 nm and at the second wavelength. As noted above for
SEC and IMAC experiments, the contribution of APols at 280 nm may have to be subtracted.
The ratio of MP and APol in the complexes can be determined from their respective extinction
coefficients.
Protocol prepared by Manuela Zoonens on the basis of Zoonens et al. (2007), Gohon et al.
(2008), Charvolin et al. (2014), and laboratory notes.
318
5 Formation and Properties of Membrane Protein/Amphipol Complexes