Fig. 9.7 Examples of size exclusion chromatography analyses of membrane protein/amphipol
complexes. (A) BR/A8-35 complexes. Arrows indicate the void (V 0 ) and total (V t ) volumes. The elution
profiles of BR/HAPol complexes (3.3 gÁL
À1 BR), BR/DAPol complexes (1.2 gÁL
À1
BR), and catalase
(220 kDa, 2 gÁL
À1
) from a Superose 12 HR column were scaled to the same maximum (From Gohon et al.
2008, # 2008 The Biophysical Society. Published by Elsevier Inc. All rights reserved). (B) Cytochrome
bc 1 /A8-35 complexes purified on sucrose gradients containing either no (À À À À) or 0.1 gÁL
-1 (——)
A8-35 were washed free of sucrose by dialysis and injected onto a Superose 6 column in surfactant-free
buffer. Elution profiles were analyzed at 220 nm (From Charvolin et al. 2014). (C) SEC analysis of
tOmpA/surfactant complexes. SEC elution profiles at various steps during the trapping procedure. An
aliquot of a sample of tOmpA (0.22 gÁL
À1
) in 6 gÁL
À1 (19.6 mM) C 8 E 4 solution was injected onto a
Superose 12 HR column equilibrated with a Tris-NaCl buffer containing the same concentration of
detergent. Another aliquot of the tOmpA/C 8 E 4 sample was supplemented with the deuterated form of
A8-35 (DAPol) in a 4:1 APol/protein mass ratio and an aliquot of the ternary tOmpA/C 8 E 4 /DAPol
complexes injected onto the column previously equilibrated with Tris-NaCl buffer containing a mixture
of C 8 E 4 and DAPol at 4 and 0.91 gÁL
À1
, respectively. A second aliquot of the tOmpA/C 8 E 4 /DAPol
complexes was incubated for 2 h with polystyrene beads, and the resulting tOmpA/DAPol complexes were
injected onto the same column, equilibrated with surfactant-free Tris-NaCl buffer (the peak at ~17 mL in
the latter profile is due to a contaminant released by the Bio-Beads). Profiles have been normalized to the
same maximum (From Zoonens et al. 2007). (D) Effect of the presence of free APols on the dispersity of
tOmpA/APol complexes. After polyhistidine-tagged tOmpA was trapped with DAPol at a 4:1 APol/
protein mass ratio, an aliquot of this sample was injected onto a Superose 12 HR column equilibrated with
Tris-NaCl buffer, pH 7.9. The rest of the sample was immobilized on a 1 mL nickel-chelating column and
free APol particles washed away with surfactant-free buffer. The tOmpA/A8-35 complexes were eluted
with 300 mM imidazole in the same buffer and desalted on a 5 mL desalting column. An aliquot of the
fraction containing tOmpA/A8-35 was then injected onto the Superose 12 HR column. The full width at
half-height (HHFW) of the peak of tOmpA/A8-35 complexes after IMAC (1.72 mL) is twice that of the
initial sample (0.84 mL). Aliquots of the aggregated sample were then supplemented with A8-35 to reach a
final overall APol/protein mass ratio of either 2:1 or 4:1. After one night of incubation, the aliquots were
injected onto the Superose 12 HR column. The HHFW values decreased to 1.36 and 0.92 mL, respectively
(Reprinted with permission from Zoonens et al. 2007, # 2007 American Chemical Society).
418
9 Solution Studies of Membrane Protein/Amphipol Complexes
complexes. (A) BR/A8-35 complexes. Arrows indicate the void (V 0 ) and total (V t ) volumes. The elution
profiles of BR/HAPol complexes (3.3 gÁL
À1 BR), BR/DAPol complexes (1.2 gÁL
À1
BR), and catalase
(220 kDa, 2 gÁL
À1
) from a Superose 12 HR column were scaled to the same maximum (From Gohon et al.
2008, # 2008 The Biophysical Society. Published by Elsevier Inc. All rights reserved). (B) Cytochrome
bc 1 /A8-35 complexes purified on sucrose gradients containing either no (À À À À) or 0.1 gÁL
-1 (——)
A8-35 were washed free of sucrose by dialysis and injected onto a Superose 6 column in surfactant-free
buffer. Elution profiles were analyzed at 220 nm (From Charvolin et al. 2014). (C) SEC analysis of
tOmpA/surfactant complexes. SEC elution profiles at various steps during the trapping procedure. An
aliquot of a sample of tOmpA (0.22 gÁL
À1
) in 6 gÁL
À1 (19.6 mM) C 8 E 4 solution was injected onto a
Superose 12 HR column equilibrated with a Tris-NaCl buffer containing the same concentration of
detergent. Another aliquot of the tOmpA/C 8 E 4 sample was supplemented with the deuterated form of
A8-35 (DAPol) in a 4:1 APol/protein mass ratio and an aliquot of the ternary tOmpA/C 8 E 4 /DAPol
complexes injected onto the column previously equilibrated with Tris-NaCl buffer containing a mixture
of C 8 E 4 and DAPol at 4 and 0.91 gÁL
À1
, respectively. A second aliquot of the tOmpA/C 8 E 4 /DAPol
complexes was incubated for 2 h with polystyrene beads, and the resulting tOmpA/DAPol complexes were
injected onto the same column, equilibrated with surfactant-free Tris-NaCl buffer (the peak at ~17 mL in
the latter profile is due to a contaminant released by the Bio-Beads). Profiles have been normalized to the
same maximum (From Zoonens et al. 2007). (D) Effect of the presence of free APols on the dispersity of
tOmpA/APol complexes. After polyhistidine-tagged tOmpA was trapped with DAPol at a 4:1 APol/
protein mass ratio, an aliquot of this sample was injected onto a Superose 12 HR column equilibrated with
Tris-NaCl buffer, pH 7.9. The rest of the sample was immobilized on a 1 mL nickel-chelating column and
free APol particles washed away with surfactant-free buffer. The tOmpA/A8-35 complexes were eluted
with 300 mM imidazole in the same buffer and desalted on a 5 mL desalting column. An aliquot of the
fraction containing tOmpA/A8-35 was then injected onto the Superose 12 HR column. The full width at
half-height (HHFW) of the peak of tOmpA/A8-35 complexes after IMAC (1.72 mL) is twice that of the
initial sample (0.84 mL). Aliquots of the aggregated sample were then supplemented with A8-35 to reach a
final overall APol/protein mass ratio of either 2:1 or 4:1. After one night of incubation, the aliquots were
injected onto the Superose 12 HR column. The HHFW values decreased to 1.36 and 0.92 mL, respectively
(Reprinted with permission from Zoonens et al. 2007, # 2007 American Chemical Society).
418
9 Solution Studies of Membrane Protein/Amphipol Complexes