9.5.2.3 Size Exclusion Chromatography System
■ For BR/amphipol complexes, SEC was performed at room temperature (RT) on an FPLC (fast
protein liquid chromatography) system (Äkta Purifier 10, GE Healthcare) equipped with a Superose
12 10/300 GL column (total volume ~20 mL). UV-visible absorbance detection was performed at three
wavelengths: 220, 280, and 554 nm. The volume of the sample loop was 100 μL. The flow rate was set
at 0.5 mL‧min
À1 .
The choice of the column must be adapted to the expected mass of MP/APol complexes. In the
case of BR/A8-35 complexes, the molar mass of the protein is 27 kDa, that of free APol particles
~40 kDa (Gohon et al. 2006; cf. Chap. 4, § 4.3.1.2), and that of BR/A8-35 complexes ~81 kDa
(Gohon et al. 2008; see Chap. 5, § 5.3.1). The separation range of the Superose 12 10/300 GL
column is 10–300 kDa.
For high-resolution separation, the volume of sample injected should be <2% of the total
column volume.
When the protein is temperature-sensitive, experiments can be carried out in a cold room.
UV-visible absorbance detection. A8-35 does not significantly absorb at 280 nm (Le Bon et al.
2014b; see Chap. 8, Fig. 8.1). The signal at 280 nm therefore is mainly due to the MP, whereas
that at 220 nm has contributions from both MP and the APol, allowing an evaluation of the
concentration of free APol in the sample (if MP/APol complexes and free APol are well
separated). Fluorescent APols (FAPols) carry covalently bound fluorescent dyes such as
fluorescein, rhodamine, NBD, Alexa Fluor 647, etc. (see Chap. 4, § 4.4, Table 4.5, and Le
Bon et al. 2014b), with absorbance peaks spread over the full visible range (see Fig. 8.9 in
Chap. 8, § 8.2.3). FAPols can be very useful tools to quantify APols present either in MP/APol
complexes or as free particles (Zoonens et al. 2014).
9.5.2.4 Buffers
■ □ Some of the experiments with BR described below were carried out in 20 mM sodium
phosphate, 100 mM NaCl, pH 7. However, this is not the optimal buffer.
When working with A8-35, do not work below pH 7, which results in polymer aggregation
(Gohon et al. 2006); it is preferable to use pH 7.5 or pH 8 buffers.
Multivalent cations, even present as traces, cause free A8-35 particles and MP/A8-35
complexes to aggregate (Picard et al. 2006; Catoire et al. 2010). SEC profiles are improved in
the presence of ~1 mM EDTA.
When working with FAPols, there can be some pH limitation to avoid dye degradation.
There is, as a rule, no need to supplement the SEC buffer with free APol. However, complete
separation of MP/APol complexes from free APol particles can result in a degree of aggregation
(Zoonens et al. 2007; see Fig. 9.7D and Chap. 5, § 5.2.1). This can be avoided by working in the
presence of some free APol (whose contribution to the absorbance profiles will have to be taken
into account when determining MP/APol ratios in complexes). Because aggregation can be
expected to depend on the protein, the buffer, etc., how much free APol to use must be
determined experimentally. Note that, when using preparative SEC, for example for cryo-EM
purposes, aggregates generated by the removal of the free APol, if any, can be dispersed by
adding back free APol to the purified complexes (Zoonens et al. 2007).
9.5 Protocols
445
■ For BR/amphipol complexes, SEC was performed at room temperature (RT) on an FPLC (fast
protein liquid chromatography) system (Äkta Purifier 10, GE Healthcare) equipped with a Superose
12 10/300 GL column (total volume ~20 mL). UV-visible absorbance detection was performed at three
wavelengths: 220, 280, and 554 nm. The volume of the sample loop was 100 μL. The flow rate was set
at 0.5 mL‧min
À1 .
The choice of the column must be adapted to the expected mass of MP/APol complexes. In the
case of BR/A8-35 complexes, the molar mass of the protein is 27 kDa, that of free APol particles
~40 kDa (Gohon et al. 2006; cf. Chap. 4, § 4.3.1.2), and that of BR/A8-35 complexes ~81 kDa
(Gohon et al. 2008; see Chap. 5, § 5.3.1). The separation range of the Superose 12 10/300 GL
column is 10–300 kDa.
For high-resolution separation, the volume of sample injected should be <2% of the total
column volume.
When the protein is temperature-sensitive, experiments can be carried out in a cold room.
UV-visible absorbance detection. A8-35 does not significantly absorb at 280 nm (Le Bon et al.
2014b; see Chap. 8, Fig. 8.1). The signal at 280 nm therefore is mainly due to the MP, whereas
that at 220 nm has contributions from both MP and the APol, allowing an evaluation of the
concentration of free APol in the sample (if MP/APol complexes and free APol are well
separated). Fluorescent APols (FAPols) carry covalently bound fluorescent dyes such as
fluorescein, rhodamine, NBD, Alexa Fluor 647, etc. (see Chap. 4, § 4.4, Table 4.5, and Le
Bon et al. 2014b), with absorbance peaks spread over the full visible range (see Fig. 8.9 in
Chap. 8, § 8.2.3). FAPols can be very useful tools to quantify APols present either in MP/APol
complexes or as free particles (Zoonens et al. 2014).
9.5.2.4 Buffers
■ □ Some of the experiments with BR described below were carried out in 20 mM sodium
phosphate, 100 mM NaCl, pH 7. However, this is not the optimal buffer.
When working with A8-35, do not work below pH 7, which results in polymer aggregation
(Gohon et al. 2006); it is preferable to use pH 7.5 or pH 8 buffers.
Multivalent cations, even present as traces, cause free A8-35 particles and MP/A8-35
complexes to aggregate (Picard et al. 2006; Catoire et al. 2010). SEC profiles are improved in
the presence of ~1 mM EDTA.
When working with FAPols, there can be some pH limitation to avoid dye degradation.
There is, as a rule, no need to supplement the SEC buffer with free APol. However, complete
separation of MP/APol complexes from free APol particles can result in a degree of aggregation
(Zoonens et al. 2007; see Fig. 9.7D and Chap. 5, § 5.2.1). This can be avoided by working in the
presence of some free APol (whose contribution to the absorbance profiles will have to be taken
into account when determining MP/APol ratios in complexes). Because aggregation can be
expected to depend on the protein, the buffer, etc., how much free APol to use must be
determined experimentally. Note that, when using preparative SEC, for example for cryo-EM
purposes, aggregates generated by the removal of the free APol, if any, can be dispersed by
adding back free APol to the purified complexes (Zoonens et al. 2007).
9.5 Protocols
445