• Protocol
5. At the end of the APol/detergent exchange, the volume of the sample is estimated with a
ruler, assuming the solution to represent ~30% of the volume of the resin (usually 5–7 mL).
Purified MSP1D1 at ~15 g‧L
À1 is added at a 40:1 MSP1D1/BLT2 molar ratio. Aliquots of
MSP1D1 (100 μL) are added to solubilized samples of DMPC (25 mM in 100 mM DDM) at
~20:1 DMPC/MSP1D1 molar ratio, final concentrations being ~4 mM DMPC and 200 μM
MSP1D1. The CHS concentration is adjusted based on the total volume of the sample after
the APol-to-detergent exchange. Different CHS/DMPC molar ratios were tested, so as to
evaluate their effect on GPCR activity (Casiraghi et al. 2016), the CHS concentration being
adjusted with 20-g‧L
À1 CHS solution in 50 mM Tris/HCl, pH 8, 100 mM DDM.
Reconstitution can be conducted either directly in the column, immediately after the
APol-to-detergent exchange, or the resin can be transferred to a glassware.
6. Once all the components are added, incubate the mixture for 1 h above the transition
temperature of the lipids (23
C in this specific case, as the transition temperature of the
binary lipid mixture was estimated to be ~20.5
C) under slight stirring in a thermostatically controlled device or a temperature-controlled room, so as to let the components mix
(Ritchie et al. 2009).
7. NDs spontaneously form upon detergent removal, for which various methods can be used
(cf. Chap. 3). If polystyrene beads are used, they must be washed several times in ethanol
followed by several more washes in Milli-Q water and finally stored in water at 4
C.
Before use, dry away the excess of water by depositing the beads on filter paper. Typically,
60% w/v Bio-Beads SM2 (Bio-Rad) are directly added to the column or vial. Stir for 2 h at
the gel-fluid phase temperature transition, then at 4
C overnight.
8. At this point, BLT2/ND complexes are separated from empty discs by IMAC. If reconstitution was performed directly in the chromatographic column, this step can be performed
by FPLC, by simply reconnecting the column to the system. For reconstitution conducted
in a glassware, the resin is poured into a disposable plastic column. In both cases, empty
discs are washed with 50 mM Tris/HCl, 150 mM NaCl, 10 mM imidazole, pH 8, after
which BLT2/ND complexes are eluted with 50 mM Tris/HCl, 150 mM NaCl, 400 mM
imidazole, pH 8.
9. Exchange the buffer of the eluate for 20 mM Tris/HCl, 100 mM NaCl, 5 mM EDTA,
pH 8, using centrifugal filters (Amicon Ultra-4, 10 kDa cutoff, Merck), and concentrate
by centrifugation at 3,500 Â g, 4
C (Allegra 64R centrifuge, Beckman Coulter), until
reaching a final volume suitable for SEC.
10. Before SEC, the sample is centrifuged at 40,000 Â g, 30 min, 4
C (Beckman Coulter,
Optima MAX-XP ultracentrifuge; rotor TLA 100), in order to remove most of the excess of
lipids, proteoliposomes, and aggregates. SEC is performed on a Superose 12 10/300 GL
column (GE Healthcare, Life Sciences) directly assembled on the Äkta purifier system
(GE Healthcare) and equilibrated with 20 mM Tris/HCl, pH 8, 100 mM NaCl, 5 mM
EDTA. The sample (~100 μL) is injected through a 100 μL loop. Typically, after reconstitution, a mixture of different particles is obtained (Fig. 5.43). The peak corresponding to
BLT2/ND complexes is pooled and characterized (usually by SDS-PAGE, EM, mass
spectrometry, etc.; see Fig. 5.44) and, if the sample is satisfying, used for NMR studies.
5.9 Protocols
321
5. At the end of the APol/detergent exchange, the volume of the sample is estimated with a
ruler, assuming the solution to represent ~30% of the volume of the resin (usually 5–7 mL).
Purified MSP1D1 at ~15 g‧L
À1 is added at a 40:1 MSP1D1/BLT2 molar ratio. Aliquots of
MSP1D1 (100 μL) are added to solubilized samples of DMPC (25 mM in 100 mM DDM) at
~20:1 DMPC/MSP1D1 molar ratio, final concentrations being ~4 mM DMPC and 200 μM
MSP1D1. The CHS concentration is adjusted based on the total volume of the sample after
the APol-to-detergent exchange. Different CHS/DMPC molar ratios were tested, so as to
evaluate their effect on GPCR activity (Casiraghi et al. 2016), the CHS concentration being
adjusted with 20-g‧L
À1 CHS solution in 50 mM Tris/HCl, pH 8, 100 mM DDM.
Reconstitution can be conducted either directly in the column, immediately after the
APol-to-detergent exchange, or the resin can be transferred to a glassware.
6. Once all the components are added, incubate the mixture for 1 h above the transition
temperature of the lipids (23
C in this specific case, as the transition temperature of the
binary lipid mixture was estimated to be ~20.5
C) under slight stirring in a thermostatically controlled device or a temperature-controlled room, so as to let the components mix
(Ritchie et al. 2009).
7. NDs spontaneously form upon detergent removal, for which various methods can be used
(cf. Chap. 3). If polystyrene beads are used, they must be washed several times in ethanol
followed by several more washes in Milli-Q water and finally stored in water at 4
C.
Before use, dry away the excess of water by depositing the beads on filter paper. Typically,
60% w/v Bio-Beads SM2 (Bio-Rad) are directly added to the column or vial. Stir for 2 h at
the gel-fluid phase temperature transition, then at 4
C overnight.
8. At this point, BLT2/ND complexes are separated from empty discs by IMAC. If reconstitution was performed directly in the chromatographic column, this step can be performed
by FPLC, by simply reconnecting the column to the system. For reconstitution conducted
in a glassware, the resin is poured into a disposable plastic column. In both cases, empty
discs are washed with 50 mM Tris/HCl, 150 mM NaCl, 10 mM imidazole, pH 8, after
which BLT2/ND complexes are eluted with 50 mM Tris/HCl, 150 mM NaCl, 400 mM
imidazole, pH 8.
9. Exchange the buffer of the eluate for 20 mM Tris/HCl, 100 mM NaCl, 5 mM EDTA,
pH 8, using centrifugal filters (Amicon Ultra-4, 10 kDa cutoff, Merck), and concentrate
by centrifugation at 3,500 Â g, 4
C (Allegra 64R centrifuge, Beckman Coulter), until
reaching a final volume suitable for SEC.
10. Before SEC, the sample is centrifuged at 40,000 Â g, 30 min, 4
C (Beckman Coulter,
Optima MAX-XP ultracentrifuge; rotor TLA 100), in order to remove most of the excess of
lipids, proteoliposomes, and aggregates. SEC is performed on a Superose 12 10/300 GL
column (GE Healthcare, Life Sciences) directly assembled on the Äkta purifier system
(GE Healthcare) and equilibrated with 20 mM Tris/HCl, pH 8, 100 mM NaCl, 5 mM
EDTA. The sample (~100 μL) is injected through a 100 μL loop. Typically, after reconstitution, a mixture of different particles is obtained (Fig. 5.43). The peak corresponding to
BLT2/ND complexes is pooled and characterized (usually by SDS-PAGE, EM, mass
spectrometry, etc.; see Fig. 5.44) and, if the sample is satisfying, used for NMR studies.
5.9 Protocols
321