• Wash the gel filtration column with three column volumes of running buffer. Note that, for
such an analysis, APol is usually not required in the running buffer, in contrast to detergents,
which must always be present above their CMC. This is also how samples for electron
microscopy (EM) are prepared before being spread out on the EM grids. However, for this
particular experiment, it cannot be excluded that a small amount of APol leaches from the
protein as the complexes migrate into APol-free buffer. It might therefore be preferable to
saturate the solution with which the column is equilibrated with “some” free APol, such as
5–10% of the concentration present in the sample, so as to prevent desorption, and to subtract
the corresponding background. However, this modification to the procedure has not been
carefully investigated yet. The composition of standard buffer is 20 mM Tris/HCl, pH 8.0,
150 mM NaCl, but it can be modified provided the pH is above 7.0 and divalent cations are
absent.
• Inject an aliquot of FAPol/A8-35 mixture (or pure FAPol) at 10 gÁL
À1 . The elution profile is
monitored at two wavelengths, 280 nm and the maximum absorbance wavelength of the
fluorophore, for example 490 nm for FAPol NBD . Determine the elution volume of APol
particles.
• After trapping the protein in the FAPol/A8-35 mixture, inject an aliquot of the sample at an
appropriate concentration in order to get a good signal-to-noise ratio of the elution peak.
Follow the elution of MP/A8-35/FAPol NBD complexes at the two wavelengths, e.g. 280 nm
and 490 nm. If the separation from free APol particles is good, calculate the amount of bound
APols per MP as follows: integrate the peak area of MP/A8-35/FAPol NBD complexes at
280 nm and 490 nm in order to determine, respectively, the mass of MP and that of FAPol NBD
which has comigrated with the protein. A subtraction of the APol contribution to the
absorbance at 280 nm may have to be applied, based on the ratio of the surface of the
peaks at 280 nm and 490 nm observed with the pure FAPol/A8-35 mixture or on the ratio of
the optical densities at 280 nm and 490 nm measured from a UV-visible spectrum. The total
mass of APol is then calculated taking into account the dilution of FAPol with A8-35. The
ratio of APol and MP masses gives the amount of bound APols per MP. (Note: if the elution
peaks of APol particles and MP/APol complexes overlap, use a more resolutive gel filtration
column or try another separation procedure.)
Method 2. Immobilized-Metal Affinity Chromatography (IMAC)
The presence of a tag fused to the MP under study makes it possible to immobilize MP/APol
complexes onto an affinity column and to eliminate the excess of APol particles. This procedure is
particularly convenient when the protein is small and MP/APol complexes cannot be efficiently
separated from free APol particles by SEC. Note that free APol particles are, however, required to
keep homogeneous MP/APol complexes. Indeed, in the absence of free APol particles, small MP/APol
oligomers tend to form, which is likely to be accompanied by some desorption of the MP-bound APol
(Zoonens et al. 2007). Because of this effect, the MP/APol ratio determined by this method must be
taken as an estimate by default unless buffers have been supplemented with some free APol.
• After MP trapping in the FAPol/A8-35 mixture, inject the sample on an affinity resin. For
instance, if the protein has a polyhistidine tag, load the sample on a Ni:NTA resin. The
majority of the protein (~80%) will be retained on the resin (Zoonens et al. 2007; Giusti et al.
2015).
• Rinse the resin with equilibration buffer to wash out free FAPol/A8-35 particles. Elute the
MP/FAPol/A8-35 complexes with a buffer containing imidazole. Note that, as noted above,
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317
such an analysis, APol is usually not required in the running buffer, in contrast to detergents,
which must always be present above their CMC. This is also how samples for electron
microscopy (EM) are prepared before being spread out on the EM grids. However, for this
particular experiment, it cannot be excluded that a small amount of APol leaches from the
protein as the complexes migrate into APol-free buffer. It might therefore be preferable to
saturate the solution with which the column is equilibrated with “some” free APol, such as
5–10% of the concentration present in the sample, so as to prevent desorption, and to subtract
the corresponding background. However, this modification to the procedure has not been
carefully investigated yet. The composition of standard buffer is 20 mM Tris/HCl, pH 8.0,
150 mM NaCl, but it can be modified provided the pH is above 7.0 and divalent cations are
absent.
• Inject an aliquot of FAPol/A8-35 mixture (or pure FAPol) at 10 gÁL
À1 . The elution profile is
monitored at two wavelengths, 280 nm and the maximum absorbance wavelength of the
fluorophore, for example 490 nm for FAPol NBD . Determine the elution volume of APol
particles.
• After trapping the protein in the FAPol/A8-35 mixture, inject an aliquot of the sample at an
appropriate concentration in order to get a good signal-to-noise ratio of the elution peak.
Follow the elution of MP/A8-35/FAPol NBD complexes at the two wavelengths, e.g. 280 nm
and 490 nm. If the separation from free APol particles is good, calculate the amount of bound
APols per MP as follows: integrate the peak area of MP/A8-35/FAPol NBD complexes at
280 nm and 490 nm in order to determine, respectively, the mass of MP and that of FAPol NBD
which has comigrated with the protein. A subtraction of the APol contribution to the
absorbance at 280 nm may have to be applied, based on the ratio of the surface of the
peaks at 280 nm and 490 nm observed with the pure FAPol/A8-35 mixture or on the ratio of
the optical densities at 280 nm and 490 nm measured from a UV-visible spectrum. The total
mass of APol is then calculated taking into account the dilution of FAPol with A8-35. The
ratio of APol and MP masses gives the amount of bound APols per MP. (Note: if the elution
peaks of APol particles and MP/APol complexes overlap, use a more resolutive gel filtration
column or try another separation procedure.)
Method 2. Immobilized-Metal Affinity Chromatography (IMAC)
The presence of a tag fused to the MP under study makes it possible to immobilize MP/APol
complexes onto an affinity column and to eliminate the excess of APol particles. This procedure is
particularly convenient when the protein is small and MP/APol complexes cannot be efficiently
separated from free APol particles by SEC. Note that free APol particles are, however, required to
keep homogeneous MP/APol complexes. Indeed, in the absence of free APol particles, small MP/APol
oligomers tend to form, which is likely to be accompanied by some desorption of the MP-bound APol
(Zoonens et al. 2007). Because of this effect, the MP/APol ratio determined by this method must be
taken as an estimate by default unless buffers have been supplemented with some free APol.
• After MP trapping in the FAPol/A8-35 mixture, inject the sample on an affinity resin. For
instance, if the protein has a polyhistidine tag, load the sample on a Ni:NTA resin. The
majority of the protein (~80%) will be retained on the resin (Zoonens et al. 2007; Giusti et al.
2015).
• Rinse the resin with equilibration buffer to wash out free FAPol/A8-35 particles. Elute the
MP/FAPol/A8-35 complexes with a buffer containing imidazole. Note that, as noted above,
5.9 Protocols
317