9.5.2.5 Protocol
1. Wash the column with 4–5 column volumes of water purified on a Millipore Milli-Q
Advantage A10 system (“Milli-Q water”).
2. Equilibrate the column by washing it with 4 column volumes of SEC buffer.
3. Inject aliquots of the MP/APols complexes prepared at the different MP/APol ratios to be
tested.
4. Inject control samples: an aliquot of an APol solution at a concentration equivalent to that
present in the MP/APols samples and aliquots of dextran (determination of the excluded/void
volume) and acetone (determination of the total volume).
The injection of an APol solution will establish the elution volume of free APols; unlabeled free
APols present in the MP/APols samples will appear at the same position in the SEC profiles
recorded at 220 nm.
5. Examination of the chromatograms will provide the following information:
• Elution volume (V el ) of MP/APols complexes: Stokes radius (see below);
• Width at half-height (WHH) and symmetry or asymmetry of the MP/APol peak: size
dispersity of the complexes and presence or not of small oligomers;
• Area under the various peaks at each wavelength: relative amounts of MP/APol complexes
and free APol particles; if using FAPols, MP/APol ratio in the complexes; in the case of
a protein like BR, whose spectrum depends on its native state, proportion of native
vs. denatured protein.
The elution volume V el of the MP/APols complexes can be correlated to their Stokes radius via
a calibration curve, by calibrating the column with standard globular proteins (which can be
obtained from GE Healthcare, Bio-Rad, etc.). See e.g. Harlan et al. (1995). Note however that
interactions between the complexes and the column may bias this evaluation (see Gohon et al.
2008 and Chap. 5, § 5.3.2).
9.5.2.6 Commented Examples
In the legends of the following three figures, a few examples of using SEC for analyzing MP/APol
samples are commented upon (Figs. 9.24, 9.25, and 9.26).
Protocol prepared by Christel Le Bon based primarily on Zoonens et al. (2007), Gohon et al.
(2008), and Le Bon et al. (2014a), and laboratory notes.
446
9 Solution Studies of Membrane Protein/Amphipol Complexes
1. Wash the column with 4–5 column volumes of water purified on a Millipore Milli-Q
Advantage A10 system (“Milli-Q water”).
2. Equilibrate the column by washing it with 4 column volumes of SEC buffer.
3. Inject aliquots of the MP/APols complexes prepared at the different MP/APol ratios to be
tested.
4. Inject control samples: an aliquot of an APol solution at a concentration equivalent to that
present in the MP/APols samples and aliquots of dextran (determination of the excluded/void
volume) and acetone (determination of the total volume).
The injection of an APol solution will establish the elution volume of free APols; unlabeled free
APols present in the MP/APols samples will appear at the same position in the SEC profiles
recorded at 220 nm.
5. Examination of the chromatograms will provide the following information:
• Elution volume (V el ) of MP/APols complexes: Stokes radius (see below);
• Width at half-height (WHH) and symmetry or asymmetry of the MP/APol peak: size
dispersity of the complexes and presence or not of small oligomers;
• Area under the various peaks at each wavelength: relative amounts of MP/APol complexes
and free APol particles; if using FAPols, MP/APol ratio in the complexes; in the case of
a protein like BR, whose spectrum depends on its native state, proportion of native
vs. denatured protein.
The elution volume V el of the MP/APols complexes can be correlated to their Stokes radius via
a calibration curve, by calibrating the column with standard globular proteins (which can be
obtained from GE Healthcare, Bio-Rad, etc.). See e.g. Harlan et al. (1995). Note however that
interactions between the complexes and the column may bias this evaluation (see Gohon et al.
2008 and Chap. 5, § 5.3.2).
9.5.2.6 Commented Examples
In the legends of the following three figures, a few examples of using SEC for analyzing MP/APol
samples are commented upon (Figs. 9.24, 9.25, and 9.26).
Protocol prepared by Christel Le Bon based primarily on Zoonens et al. (2007), Gohon et al.
(2008), and Le Bon et al. (2014a), and laboratory notes.
446
9 Solution Studies of Membrane Protein/Amphipol Complexes