• Calculate the appropriate amount of beads according to the mass of detergent present in each
sample. Weigh the beads and add them in the five samples containing APols and in one of the
two APol-free control samples, which will become the negative control (detergent removal in
the absence of APol). Note: Bio-Beads are usually washed out successively in ethanol and
water prior to use and then stored in water. Before weighing, drop them off on a tissue paper
for maximal water removal. The last sample, without beads, represents the positive control
(MP kept in detergent solution).
• Incubate the samples for 2 h under gentle shaking at either room temperature or 4
C.
• Remove the beads by pipetting the samples while excluding the beads. For that, apply the tip
of the micropipette flush with the wall of the Eppendorf tube so that only the solution is
sucked in. Put the samples in new Eppendorf tubes.
Alternatively, it is possible to eliminate the detergent micelles by dilution under the CMC of the
detergent. Note that this method is more suitable to detergents with a high CMC rather than detergents
with a low one, such as n-dodecyl-β-D-maltoside (DDM), because even under the CMC these
detergents are still able to keep MPs soluble. If the dilution method is employed, dilute the five
samples containing APols plus that of the negative control with a detergent-free buffer. Dilute the last
sample, which becomes the positive control, with buffer containing detergent at the same concentration
as initially present in the sample.
Whatever the protocol used, some detergent monomers can still be present in the samples.
Usually, they are not problematic, as long as the negative control shows that the monomers cannot
keep the MP in solution in the absence of APols, but if need be they can be eliminated by dialysis or by
several cycles of dilution/concentration using ultrafiltration devices. Note that the presence of APols in
the external dialysis buffer is not required as APols do not cross standard dialysis membranes of
12–14 kDa MW cutoff. Indeed, the MW of the particles of A8-35 is ~40 kDa (Gohon et al. 2006), and,
because of its low CAC (~0.002 gÁL
À1 ) (Giusti et al. 2012), there are very few free molecules in
solution (see Chap. 4, § 4.3.1.1). Another procedure for detergent removal, albeit seldom used, is to
adsorb it onto cyclodextrins (Althoff et al. 2011).
5.9.1.5 Identification of the Optimal MP/APol Ratio
• Measure the UV-visible spectrum of each sample.
• Centrifuge the samples at 100,000 Â g for 20 min. (Note: the speed and duration of the
centrifugation step are given for a small protein of ~30 kDa. These parameters may have to be
adjusted if the protein of interest is larger so as to make sure that close to 100% of the protein
remains in the supernatant in the presence of detergent.)
• Take off the supernatants and measure again their UV-visible spectra.
• Calculate the percentage of protein kept in the supernatant for each condition. This experiment determines the minimal MP/APol mass ratio required to keep !90% of the MP soluble
(Fig. 5.42). However, to establish the minimal MP/APol mass ratio required to obtain
homogeneous complexes, which is somewhat higher, it is recommended to analyze the
samples by SEC (cf. Chap. 9, § 9.5, Protocol 9.2).
The optimal MP/APol mass ratios for two model MPs of small MW like bacteriorhodopsin of
H. salinarum (BR, 27 kDa) and the transmembrane domain of OmpA of E. coli (tOmpA, 19 kDa) are
1:5 and 1:4, respectively (Zoonens et al. 2007; Gohon et al. 2008). These ratios exceed by !2Â the
amount of A8-35 that binds to these MPs (see Protocol 5.3). This is because APols, which have a weak
dissociating power, cannot prevent protein/protein interaction if they are not present in excess in the
sample (see § 5.2.1). To keep MP/APol complexes homogeneously distributed, an excess of APols is
5.9 Protocols
313
sample. Weigh the beads and add them in the five samples containing APols and in one of the
two APol-free control samples, which will become the negative control (detergent removal in
the absence of APol). Note: Bio-Beads are usually washed out successively in ethanol and
water prior to use and then stored in water. Before weighing, drop them off on a tissue paper
for maximal water removal. The last sample, without beads, represents the positive control
(MP kept in detergent solution).
• Incubate the samples for 2 h under gentle shaking at either room temperature or 4
C.
• Remove the beads by pipetting the samples while excluding the beads. For that, apply the tip
of the micropipette flush with the wall of the Eppendorf tube so that only the solution is
sucked in. Put the samples in new Eppendorf tubes.
Alternatively, it is possible to eliminate the detergent micelles by dilution under the CMC of the
detergent. Note that this method is more suitable to detergents with a high CMC rather than detergents
with a low one, such as n-dodecyl-β-D-maltoside (DDM), because even under the CMC these
detergents are still able to keep MPs soluble. If the dilution method is employed, dilute the five
samples containing APols plus that of the negative control with a detergent-free buffer. Dilute the last
sample, which becomes the positive control, with buffer containing detergent at the same concentration
as initially present in the sample.
Whatever the protocol used, some detergent monomers can still be present in the samples.
Usually, they are not problematic, as long as the negative control shows that the monomers cannot
keep the MP in solution in the absence of APols, but if need be they can be eliminated by dialysis or by
several cycles of dilution/concentration using ultrafiltration devices. Note that the presence of APols in
the external dialysis buffer is not required as APols do not cross standard dialysis membranes of
12–14 kDa MW cutoff. Indeed, the MW of the particles of A8-35 is ~40 kDa (Gohon et al. 2006), and,
because of its low CAC (~0.002 gÁL
À1 ) (Giusti et al. 2012), there are very few free molecules in
solution (see Chap. 4, § 4.3.1.1). Another procedure for detergent removal, albeit seldom used, is to
adsorb it onto cyclodextrins (Althoff et al. 2011).
5.9.1.5 Identification of the Optimal MP/APol Ratio
• Measure the UV-visible spectrum of each sample.
• Centrifuge the samples at 100,000 Â g for 20 min. (Note: the speed and duration of the
centrifugation step are given for a small protein of ~30 kDa. These parameters may have to be
adjusted if the protein of interest is larger so as to make sure that close to 100% of the protein
remains in the supernatant in the presence of detergent.)
• Take off the supernatants and measure again their UV-visible spectra.
• Calculate the percentage of protein kept in the supernatant for each condition. This experiment determines the minimal MP/APol mass ratio required to keep !90% of the MP soluble
(Fig. 5.42). However, to establish the minimal MP/APol mass ratio required to obtain
homogeneous complexes, which is somewhat higher, it is recommended to analyze the
samples by SEC (cf. Chap. 9, § 9.5, Protocol 9.2).
The optimal MP/APol mass ratios for two model MPs of small MW like bacteriorhodopsin of
H. salinarum (BR, 27 kDa) and the transmembrane domain of OmpA of E. coli (tOmpA, 19 kDa) are
1:5 and 1:4, respectively (Zoonens et al. 2007; Gohon et al. 2008). These ratios exceed by !2Â the
amount of A8-35 that binds to these MPs (see Protocol 5.3). This is because APols, which have a weak
dissociating power, cannot prevent protein/protein interaction if they are not present in excess in the
sample (see § 5.2.1). To keep MP/APol complexes homogeneously distributed, an excess of APols is
5.9 Protocols
313