detergents to keep MPs soluble and endow them with a charge proportional to the amount of bound
dye. Upon electrophoresis on a polyacrylamide gel in non-denaturing conditions, MP/CBB complexes
migrate as a function of their molecular mass, which can be roughly estimated, and of their charge,
essentially carried by CBB. Following migration, their activity can be assayed in the gel (Schägger and
von Jagow 1991).
BN-PAGE can be readily applied to APol-trapped MP complexes (Table 9.1). In Study 9.18, it
was used to identify mitochondrial supercomplexes fractionated in sucrose gradients either in
digitonin-solubilized form (Fig. 9.11, left) or after trapping with A8-35 (right). Other studies of either
A8-35- or NAPol-trapped MPs or MP complexes have also resorted to BN-PAGE (Table 9.1). At the
time of this writing, it has not been examined to which extent CBB mixes with APols or totally
displaces them. However, the general similarity in the position of the bands in the gels on the left and
right sides of Fig. 9.11, which correspond, respectively, to digitonin-solubilized and to A8-35-trapped
supercomplexes, tends to suggest that displacement is more or less complete.
9.3.4
Chemical Cross-Linking
Trapping with APols does not prevent studying protein-protein interactions by chemical cross-linking
either with glutaraldehyde (Studies 9.2 and 9.3) or with disuccinimidyl tartrate (Study 9.30).
9.3.5
Electron Resonance Spectroscopy
In Study 9.24, double electron-electron resonance (DEER) spectroscopy was applied to measure the
distance between two nitroxide-labeled positions in SMA-trapped BR, so as to compare it with
distances measured in BR crystals.
Fig. 9.10 Size exclusion chromatography analysis of ExbB 4 /ExbD 2 complexes. Left. Analytical SEC of
the DDM-solubilized or A8-35-trapped complexes on a Superose 6 SEC column. Right. Independent
measurements of the Stokes radius R S by asymmetric flow field-flow fractionation-multi-angle laser light
scattering (AF4-MALLS) are consistent with the R H of protein/APol complexes determined by SEC
(From Sverzhinsky et al. 2014).
9.3 A Walk Through Selected Solution Studies
421
dye. Upon electrophoresis on a polyacrylamide gel in non-denaturing conditions, MP/CBB complexes
migrate as a function of their molecular mass, which can be roughly estimated, and of their charge,
essentially carried by CBB. Following migration, their activity can be assayed in the gel (Schägger and
von Jagow 1991).
BN-PAGE can be readily applied to APol-trapped MP complexes (Table 9.1). In Study 9.18, it
was used to identify mitochondrial supercomplexes fractionated in sucrose gradients either in
digitonin-solubilized form (Fig. 9.11, left) or after trapping with A8-35 (right). Other studies of either
A8-35- or NAPol-trapped MPs or MP complexes have also resorted to BN-PAGE (Table 9.1). At the
time of this writing, it has not been examined to which extent CBB mixes with APols or totally
displaces them. However, the general similarity in the position of the bands in the gels on the left and
right sides of Fig. 9.11, which correspond, respectively, to digitonin-solubilized and to A8-35-trapped
supercomplexes, tends to suggest that displacement is more or less complete.
9.3.4
Chemical Cross-Linking
Trapping with APols does not prevent studying protein-protein interactions by chemical cross-linking
either with glutaraldehyde (Studies 9.2 and 9.3) or with disuccinimidyl tartrate (Study 9.30).
9.3.5
Electron Resonance Spectroscopy
In Study 9.24, double electron-electron resonance (DEER) spectroscopy was applied to measure the
distance between two nitroxide-labeled positions in SMA-trapped BR, so as to compare it with
distances measured in BR crystals.
Fig. 9.10 Size exclusion chromatography analysis of ExbB 4 /ExbD 2 complexes. Left. Analytical SEC of
the DDM-solubilized or A8-35-trapped complexes on a Superose 6 SEC column. Right. Independent
measurements of the Stokes radius R S by asymmetric flow field-flow fractionation-multi-angle laser light
scattering (AF4-MALLS) are consistent with the R H of protein/APol complexes determined by SEC
(From Sverzhinsky et al. 2014).
9.3 A Walk Through Selected Solution Studies
421