and injected onto a SEC column in surfactant-free buffer. The complexes purified in the presence of
free APol migrated as reasonably monodisperse particles, whereas those depleted of it contained small
aggregates, mainly dimers (Charvolin et al. 2014).
Panel C, from Study 9.9, compares the SEC profile of tOmpA solubilized in the detergent C 8 E 4 ,
of the same preparation supplemented with A8-35, and finally after depletion of C 8 E 4 by adsorption
onto polystyrene beads. One notes that tOmpA/A8-35 complexes elute both earlier and as a broader
band than tOmpA/C 8 E 4 ones, whereas the ternary tOmpA/A8-35/C 8 E 4 complexes appear as large as
the tOmpA/A8-35 ones but significantly more monodisperse. The origin of this phenomenon and its
potential usefulness for crystallization attempts will be discussed in Chap. 11, § 11.3.1.
Finally, Panel D, also from Study 9.9, illustrates the reversibility of the aggregation induced by
free APol depletion: an aliquot from a preparation of polyhistidine-tagged tOmpA trapped in A8-35 at
a 1:4 protein/APol mass ratio was analyzed by SEC, and the rest of the sample separated from free
A8-35 by immobilized metal affinity chromatography (IMAC). An aliquot of the resulting preparation,
examined by SEC, exhibited a broad polydispersity, indicative of the formation of small aggregates.
Two fractions from the depleted sample were supplemented with A8-35 so as to reach the final mass
ratios of, respectively, 2 or 4 g of A8-35 per g tOmpA. SEC analysis revealed that, at 2 g per g tOmpA,
aggregation was partially reversed, whereas at 4 g per g tOmpA, the reversal was essentially complete
(Zoonens et al. 2007).
Other commented examples of SEC profiles are shown in § 9.5.2, Figs. 9.24, 9.25, and 9.26.
SEC has been exploited to examine the effect of the presence of divalent cations in buffers on
the dispersity of the particles formed by various APols and, as a consequence, on that of the complexes
formed between these APols and MPs (Study 9.10). A comparison is shown in Fig. 9.8 of the behavior
of A8-35, SAPol, PMAL-C12, and PMALA-C12 particles in the presence or absence of low
concentrations of Ca
2+ and/or Mg
2+ . The distribution of the particles of A8-35 (from a rather poor
batch, which formed heterogeneous particles) is slightly sensitive to the presence of 1 mM Mg
2+ ,
which increases the heterogeneity, and much more strongly affected by that of 1 mM Mg
2+ + 0.5 mM
Ca
2+ , which entails an important degree of aggregation (Fig. 9.8A). The size and dispersity of particles
of a sulfonated APol (SAPol; see Dahmane et al. 2011; formula shown in Chap. 4, Fig. 4.1) are
essentially insensitive to either condition (Fig. 9.8B). The particles of PMAL-C12, a zwitterionic APol
(Nagy et al. 2001; ibid.), are larger than those of A8-35 but behave essentially like them upon addition
of either Mg
2+ or Mg
2+ + Ca
2+ (Fig. 9.8C). Finally, the particles of PMALA-C12, a sulfonated,
zwitterionic APol with a net negative charge at the pH of the experiment, appear extremely large and
polydisperse even in the absence of divalent cations, and their dispersity seems rather insensitive to
their addition (Fig. 9.8D).
In addition to MP/A8-35 and MP/SAPol complexes (see above and Table 9.1), MP/polymer
complexes that have been studied by SEC include those with non-ionic APols (Study 9.14, etc.),
PMAL-C8 (Study 9.46), SMA (Study 9.17, etc.), DIBMA (Study 9.58), NVoy (Study 9.21;
cf. Fig. 9.9), amphipathic derivatives of poly-γ-glutamic acid (Studies 9.36 and 9.55), as well as a
variety of labeled or tagged versions of A8-35 (see further references in Table 9.1).
Analytical SEC can be combined with various optical techniques, as well as with the use of
labeled APols, in order to determine the respective contributions of the protein and polymer to the mass
of the particles (Fig. 9.9) or the Stokes radius of the complexes (Fig. 9.10).
9.3.3
Blue-Native Polyacrylamide Gel Electrophoresis
Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) relies on supplementing MPs or
MP complexes with the amphipathic dye Coomassie Brilliant Blue (CBB), which can substitute
9.3 A Walk Through Selected Solution Studies
419
free APol migrated as reasonably monodisperse particles, whereas those depleted of it contained small
aggregates, mainly dimers (Charvolin et al. 2014).
Panel C, from Study 9.9, compares the SEC profile of tOmpA solubilized in the detergent C 8 E 4 ,
of the same preparation supplemented with A8-35, and finally after depletion of C 8 E 4 by adsorption
onto polystyrene beads. One notes that tOmpA/A8-35 complexes elute both earlier and as a broader
band than tOmpA/C 8 E 4 ones, whereas the ternary tOmpA/A8-35/C 8 E 4 complexes appear as large as
the tOmpA/A8-35 ones but significantly more monodisperse. The origin of this phenomenon and its
potential usefulness for crystallization attempts will be discussed in Chap. 11, § 11.3.1.
Finally, Panel D, also from Study 9.9, illustrates the reversibility of the aggregation induced by
free APol depletion: an aliquot from a preparation of polyhistidine-tagged tOmpA trapped in A8-35 at
a 1:4 protein/APol mass ratio was analyzed by SEC, and the rest of the sample separated from free
A8-35 by immobilized metal affinity chromatography (IMAC). An aliquot of the resulting preparation,
examined by SEC, exhibited a broad polydispersity, indicative of the formation of small aggregates.
Two fractions from the depleted sample were supplemented with A8-35 so as to reach the final mass
ratios of, respectively, 2 or 4 g of A8-35 per g tOmpA. SEC analysis revealed that, at 2 g per g tOmpA,
aggregation was partially reversed, whereas at 4 g per g tOmpA, the reversal was essentially complete
(Zoonens et al. 2007).
Other commented examples of SEC profiles are shown in § 9.5.2, Figs. 9.24, 9.25, and 9.26.
SEC has been exploited to examine the effect of the presence of divalent cations in buffers on
the dispersity of the particles formed by various APols and, as a consequence, on that of the complexes
formed between these APols and MPs (Study 9.10). A comparison is shown in Fig. 9.8 of the behavior
of A8-35, SAPol, PMAL-C12, and PMALA-C12 particles in the presence or absence of low
concentrations of Ca
2+ and/or Mg
2+ . The distribution of the particles of A8-35 (from a rather poor
batch, which formed heterogeneous particles) is slightly sensitive to the presence of 1 mM Mg
2+ ,
which increases the heterogeneity, and much more strongly affected by that of 1 mM Mg
2+ + 0.5 mM
Ca
2+ , which entails an important degree of aggregation (Fig. 9.8A). The size and dispersity of particles
of a sulfonated APol (SAPol; see Dahmane et al. 2011; formula shown in Chap. 4, Fig. 4.1) are
essentially insensitive to either condition (Fig. 9.8B). The particles of PMAL-C12, a zwitterionic APol
(Nagy et al. 2001; ibid.), are larger than those of A8-35 but behave essentially like them upon addition
of either Mg
2+ or Mg
2+ + Ca
2+ (Fig. 9.8C). Finally, the particles of PMALA-C12, a sulfonated,
zwitterionic APol with a net negative charge at the pH of the experiment, appear extremely large and
polydisperse even in the absence of divalent cations, and their dispersity seems rather insensitive to
their addition (Fig. 9.8D).
In addition to MP/A8-35 and MP/SAPol complexes (see above and Table 9.1), MP/polymer
complexes that have been studied by SEC include those with non-ionic APols (Study 9.14, etc.),
PMAL-C8 (Study 9.46), SMA (Study 9.17, etc.), DIBMA (Study 9.58), NVoy (Study 9.21;
cf. Fig. 9.9), amphipathic derivatives of poly-γ-glutamic acid (Studies 9.36 and 9.55), as well as a
variety of labeled or tagged versions of A8-35 (see further references in Table 9.1).
Analytical SEC can be combined with various optical techniques, as well as with the use of
labeled APols, in order to determine the respective contributions of the protein and polymer to the mass
of the particles (Fig. 9.9) or the Stokes radius of the complexes (Fig. 9.10).
9.3.3
Blue-Native Polyacrylamide Gel Electrophoresis
Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) relies on supplementing MPs or
MP complexes with the amphipathic dye Coomassie Brilliant Blue (CBB), which can substitute
9.3 A Walk Through Selected Solution Studies
419