5 M stock solution), 5 mM MgOAc 2 (5 mL of 1 M stock
solution), 10% glycerol.
4. cOmplete protease inhibitor cocktail tablets, EDTA-free
(Roche).
5. Prepare 100 mM PMSF (phenylmethylsulfonyl fluoride) stock
solution: add 17.4 mg of PMSF per 1 mL of isopropanol and
freeze 20 μL aliquots and store at À20
C.
6. Ultracentrifuge, Ti70 rotor, and centrifugation tubes (Beckman Coulter).
7. Dounce homogenizer.
2.3 Detergent
Solubilization
1. Prepare 10% w/v DDM (n-dodecyl-β-D-maltoside, Anatrace)
stock solution in water. Dissolve by gently agitating; do not
vortex.
2. Head-over-tail rotator or equivalent.
3. Ultracentrifuge, Ti70 rotor, and centrifugation tubes (Beckman Coulter).
2.4 NiNTA Affinity
Chromatography
1. Ni-NTA agarose resin (Qiagen).
2. Gravity flow column (e.g., MoBiTec, #10141).
3. Prepare 100 mL Ni-NTA buffer: 20 mM HEPES-KOH
pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl
(2.6 mL of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of
1 M stock solution), 10% glycerol, 10 mM imidazole (1 mL of
1 M stock solution, adjusted to pH 8.0), 0.1% DDM (1 mL of
10% stock solution).
4. Prepare 50 mL high salt buffer: 20 mM HEPES-KOH pH 8.0
(1 mL of 1 M stock solution), 500 mM NaCl (5 mL of 5 M
stock solution), 5 mM MgOAc 2 (250 μL of 1 M stock solution), 10% glycerol, 10 mM imidazole (0.5 mL of 1 M stock
solution, adjusted to pH 8.0), 0.1% DDM (0.5 mL of 10%
stock solution).
5. Prepare 100 mL Ni-NTA Wash buffer: 20 mM HEPES-KOH
pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of 1 M stock
solution), 10% glycerol, 50 mM imidazole (5 mL of 1 M stock
solution, adjusted to pH 8.0), 0.1% DDM (1 mL of 10% stock
solution).
6. Prepare 100 mL Ni-NTA elution buffer: 20 mM HEPESKOH pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl
(2.6 mL of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of
1 M stock solution), 10% glycerol, 300 mM imidazole (30 mL
of 1 M stock solution, adjusted to pH 8.0), 0.1% DDM (1 mL
of 10% stock solution).
7. Nanodrop spectrophotometer (Thermo Scientific).
Production of Multi-subunit Membrane Protein Complexes
9
solution), 10% glycerol.
4. cOmplete protease inhibitor cocktail tablets, EDTA-free
(Roche).
5. Prepare 100 mM PMSF (phenylmethylsulfonyl fluoride) stock
solution: add 17.4 mg of PMSF per 1 mL of isopropanol and
freeze 20 μL aliquots and store at À20
C.
6. Ultracentrifuge, Ti70 rotor, and centrifugation tubes (Beckman Coulter).
7. Dounce homogenizer.
2.3 Detergent
Solubilization
1. Prepare 10% w/v DDM (n-dodecyl-β-D-maltoside, Anatrace)
stock solution in water. Dissolve by gently agitating; do not
vortex.
2. Head-over-tail rotator or equivalent.
3. Ultracentrifuge, Ti70 rotor, and centrifugation tubes (Beckman Coulter).
2.4 NiNTA Affinity
Chromatography
1. Ni-NTA agarose resin (Qiagen).
2. Gravity flow column (e.g., MoBiTec, #10141).
3. Prepare 100 mL Ni-NTA buffer: 20 mM HEPES-KOH
pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl
(2.6 mL of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of
1 M stock solution), 10% glycerol, 10 mM imidazole (1 mL of
1 M stock solution, adjusted to pH 8.0), 0.1% DDM (1 mL of
10% stock solution).
4. Prepare 50 mL high salt buffer: 20 mM HEPES-KOH pH 8.0
(1 mL of 1 M stock solution), 500 mM NaCl (5 mL of 5 M
stock solution), 5 mM MgOAc 2 (250 μL of 1 M stock solution), 10% glycerol, 10 mM imidazole (0.5 mL of 1 M stock
solution, adjusted to pH 8.0), 0.1% DDM (0.5 mL of 10%
stock solution).
5. Prepare 100 mL Ni-NTA Wash buffer: 20 mM HEPES-KOH
pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of 1 M stock
solution), 10% glycerol, 50 mM imidazole (5 mL of 1 M stock
solution, adjusted to pH 8.0), 0.1% DDM (1 mL of 10% stock
solution).
6. Prepare 100 mL Ni-NTA elution buffer: 20 mM HEPESKOH pH 8.0 (2 mL of 1 M stock solution), 130 mM NaCl
(2.6 mL of 5 M stock solution), 5 mM MgOAc 2 (0.5 mL of
1 M stock solution), 10% glycerol, 300 mM imidazole (30 mL
of 1 M stock solution, adjusted to pH 8.0), 0.1% DDM (1 mL
of 10% stock solution).
7. Nanodrop spectrophotometer (Thermo Scientific).
Production of Multi-subunit Membrane Protein Complexes
9