biomedical applications, including vaccination, using APols as a delivery vector for MPs or hydrophobic peptides, and so forth (Chap. 15).
5.9
Protocols
Three standard protocols are proposed below:
• Protocol 5.1. Transferring a membrane protein from a detergent to an amphipol environment.
• Protocol 5.2. Measuring the amount of protein-bound APol.
• Protocol 5.3. Transferring an APol-trapped MP to nanodiscs.
Comments are printed in italics and indicated by a pointing hand ( ), the protocols themselves
being printed in roman.
5.9.1
Protocol 5.1. Transferring MPs from Detergents to APols
Even though direct extraction of MPs from biological membranes by APols has been observed
(cf. Box 5.1), detergents are usually resorted to at the solubilization step, unless MPs are produced by
cell-free expression system (Chap. 7) or folded directly in the presence of APols (Chap. 6). The transfer
procedure consists in replacing detergents by APols in a sample of MPs, which, in general, but not
necessarily, has already been purified. The protocol is simple, easy, fast, and requires no important
biochemical optimization.
5.9.1.1 Preparation of a Stock Solution of APols
A8-35 is supplied as a white powder, which can be stored at room temperature. Note that most APols
are very stable molecules, except for glucosylated NAPols and FAPols, which carry sugar groups and
fluorescent probes, respectively. Storage of glucosylated NAPols at À20
C is advisable whatever their
conditioning, i.e. in powder or in solution, because sugars can be hydrolyzed. FAPols need to be
protected from UV-visible light with aluminum foil. When needed, a stock solution of APols at
100 gÁL
À1 , or 10% w/w, is prepared with Milli-Q water (water purified on an A10 Advantage Millipore
system):
• Weigh some powder, for instance 20 mg, with an analytical balance in an Eppendorf tube or a
small glass vial. (Note: the powder is sometimes very electrostatic and caution is required.)
• Add 180 μL of Milli-Q water in order to reach a final mass of 200 mg.
• Homogenize the solution with a vortex or by magnetic stirring. Incubate at least a couple of
hours before use for a good rehydration and dispersion of the polymer. The solution is then
kept at 4
C or, if need be, stored frozen at À20
C.
5.9.1.2 Determination of the Protein Concentration
The exchange of detergent for APols is carried out by supplying APols pre-solubilized in water to the
sample of MPs. The amount of APol to add is calculated on the basis of the mass of MP present in the
sample. The concentration of protein must therefore be known, at least approximately. It can be
determined by its optical density from UV-visible spectra. If the epsilon coefficient of the protein is
5.9 Protocols
311
5.9
Protocols
Three standard protocols are proposed below:
• Protocol 5.1. Transferring a membrane protein from a detergent to an amphipol environment.
• Protocol 5.2. Measuring the amount of protein-bound APol.
• Protocol 5.3. Transferring an APol-trapped MP to nanodiscs.
Comments are printed in italics and indicated by a pointing hand ( ), the protocols themselves
being printed in roman.
5.9.1
Protocol 5.1. Transferring MPs from Detergents to APols
Even though direct extraction of MPs from biological membranes by APols has been observed
(cf. Box 5.1), detergents are usually resorted to at the solubilization step, unless MPs are produced by
cell-free expression system (Chap. 7) or folded directly in the presence of APols (Chap. 6). The transfer
procedure consists in replacing detergents by APols in a sample of MPs, which, in general, but not
necessarily, has already been purified. The protocol is simple, easy, fast, and requires no important
biochemical optimization.
5.9.1.1 Preparation of a Stock Solution of APols
A8-35 is supplied as a white powder, which can be stored at room temperature. Note that most APols
are very stable molecules, except for glucosylated NAPols and FAPols, which carry sugar groups and
fluorescent probes, respectively. Storage of glucosylated NAPols at À20
C is advisable whatever their
conditioning, i.e. in powder or in solution, because sugars can be hydrolyzed. FAPols need to be
protected from UV-visible light with aluminum foil. When needed, a stock solution of APols at
100 gÁL
À1 , or 10% w/w, is prepared with Milli-Q water (water purified on an A10 Advantage Millipore
system):
• Weigh some powder, for instance 20 mg, with an analytical balance in an Eppendorf tube or a
small glass vial. (Note: the powder is sometimes very electrostatic and caution is required.)
• Add 180 μL of Milli-Q water in order to reach a final mass of 200 mg.
• Homogenize the solution with a vortex or by magnetic stirring. Incubate at least a couple of
hours before use for a good rehydration and dispersion of the polymer. The solution is then
kept at 4
C or, if need be, stored frozen at À20
C.
5.9.1.2 Determination of the Protein Concentration
The exchange of detergent for APols is carried out by supplying APols pre-solubilized in water to the
sample of MPs. The amount of APol to add is calculated on the basis of the mass of MP present in the
sample. The concentration of protein must therefore be known, at least approximately. It can be
determined by its optical density from UV-visible spectra. If the epsilon coefficient of the protein is
5.9 Protocols
311