of counterions could be exploited to distinguish MPs from soluble proteins in a rough detergent extract
that has been trapped in polyanionic APols or to prevent comigration of proteins that bind or not APols.
Quantitatively, running SGSV or AUC experiments in the presence of various alkaline cations could
possibly provide a way to determine the mass of APol bound to a given MP and, therefore, the mass of
the latter, without having to resort to labeled APols. This however would require assessing the extent to
which this substitution may affect the amount of MP-bound APol and/or its hydration and, therefore,
the mass and hydrodynamic radius of the particles. As discussed in Chap. 12 (§ 12.3.4.3), substituting
Li
+ or Na
+ counterions with Cs
+ ones could also have interesting applications in electron
cryomicroscopy.
9.3.2
Size Exclusion Chromatography
Size exclusion chromatography (SEC) relies on filtrating particles on a column of hollow beads, which
offer a larger accessible volume of solution for small than for large particles to diffuse in. As a result,
particles elute as a function of their hydrodynamic radius R H , large ones eluting more rapidly than
small ones. R H estimates are obtained by comparing the elution position of a particle to those of a set of
soluble globular molecules of known R H (see e.g. Harlan et al. 1995). SEC is by far the most
commonly used technique for characterizing the size and homogeneity of MP/APol complexes
(Table 9.1).
Some typical examples are gathered in Fig. 9.6, each of which offers some specific interest. In
Panel A, the elution pattern of BR/A8-35 complexes is compared to that of catalase, a soluble, globular
protein with a molecular mass of 220 kDa. This panel illustrates several interesting points:
(i) When BR was trapped with the batch of A8-35 denoted “HAPol” (a plain, unlabeled batch),
the complexes migrated with the same apparent size and dispersity as catalase, indicative of
their homogeneity.
(ii) Their size is overestimated. Indeed, other experiments lead to conclude that the mass of the
particles is actually close to 90 kDa, not to 220 kDa (see § 9.3.8.2). The origin of this effect
is not clear, as it does not seem attributable to electrostatic repulsion between the column
and the particles despite the relatively low ionic strength of the buffers used (typically
100–150 mM NaCl) (for a discussion, see § 9.3.8.2 and Zoonens et al. 2007).
(iii) When BR was trapped with the batch denoted “DAPol” (a partially deuterated batch), a
degree of aggregation is perceptible, revealed by the shoulder that precedes the main peak.
This was confirmed by AUC analyses (see Fig. 9.13). The formation of small oligomers is
unrelated to the deuteration. It results from the fact that, due to problems that arose during
the synthesis, this particular batch of A8-35 ended up being more hydrophobic than had
been aimed for (see Chap. 4, § 4.3.1.2.2). This is a reminder that, whereas the synthesis of
A8-35 may seem simple on paper, it has to be carefully controlled if optimal results are to be
obtained (see Chap. 4, § 4.5, Protocol 4.1).
Panel B, from Study 9.7, provides one example of the effect of depleting a MP/APol preparation
of free APol. Cytochrome bc 1 /A8-35 complexes were purified on sucrose gradients containing either
no or 0.1 gÁL
À1 A8-35. The fractions containing the complexes were pooled, washed free of sucrose,
9.3 A Walk Through Selected Solution Studies
417
that has been trapped in polyanionic APols or to prevent comigration of proteins that bind or not APols.
Quantitatively, running SGSV or AUC experiments in the presence of various alkaline cations could
possibly provide a way to determine the mass of APol bound to a given MP and, therefore, the mass of
the latter, without having to resort to labeled APols. This however would require assessing the extent to
which this substitution may affect the amount of MP-bound APol and/or its hydration and, therefore,
the mass and hydrodynamic radius of the particles. As discussed in Chap. 12 (§ 12.3.4.3), substituting
Li
+ or Na
+ counterions with Cs
+ ones could also have interesting applications in electron
cryomicroscopy.
9.3.2
Size Exclusion Chromatography
Size exclusion chromatography (SEC) relies on filtrating particles on a column of hollow beads, which
offer a larger accessible volume of solution for small than for large particles to diffuse in. As a result,
particles elute as a function of their hydrodynamic radius R H , large ones eluting more rapidly than
small ones. R H estimates are obtained by comparing the elution position of a particle to those of a set of
soluble globular molecules of known R H (see e.g. Harlan et al. 1995). SEC is by far the most
commonly used technique for characterizing the size and homogeneity of MP/APol complexes
(Table 9.1).
Some typical examples are gathered in Fig. 9.6, each of which offers some specific interest. In
Panel A, the elution pattern of BR/A8-35 complexes is compared to that of catalase, a soluble, globular
protein with a molecular mass of 220 kDa. This panel illustrates several interesting points:
(i) When BR was trapped with the batch of A8-35 denoted “HAPol” (a plain, unlabeled batch),
the complexes migrated with the same apparent size and dispersity as catalase, indicative of
their homogeneity.
(ii) Their size is overestimated. Indeed, other experiments lead to conclude that the mass of the
particles is actually close to 90 kDa, not to 220 kDa (see § 9.3.8.2). The origin of this effect
is not clear, as it does not seem attributable to electrostatic repulsion between the column
and the particles despite the relatively low ionic strength of the buffers used (typically
100–150 mM NaCl) (for a discussion, see § 9.3.8.2 and Zoonens et al. 2007).
(iii) When BR was trapped with the batch denoted “DAPol” (a partially deuterated batch), a
degree of aggregation is perceptible, revealed by the shoulder that precedes the main peak.
This was confirmed by AUC analyses (see Fig. 9.13). The formation of small oligomers is
unrelated to the deuteration. It results from the fact that, due to problems that arose during
the synthesis, this particular batch of A8-35 ended up being more hydrophobic than had
been aimed for (see Chap. 4, § 4.3.1.2.2). This is a reminder that, whereas the synthesis of
A8-35 may seem simple on paper, it has to be carefully controlled if optimal results are to be
obtained (see Chap. 4, § 4.5, Protocol 4.1).
Panel B, from Study 9.7, provides one example of the effect of depleting a MP/APol preparation
of free APol. Cytochrome bc 1 /A8-35 complexes were purified on sucrose gradients containing either
no or 0.1 gÁL
À1 A8-35. The fractions containing the complexes were pooled, washed free of sucrose,
9.3 A Walk Through Selected Solution Studies
417