8. Discard the supernatant and resuspend the brown-yellow pellet
(membranes) in ~5 mL HSGM buffer using a Dounce homogenizer. Add cOmplete EDTA-free protease inhibitor cocktail
and 0.5 mM PMSF (see Note 4).
9. Analyze the membrane fraction using a 15% acrylamide gel to
confirm the presence of SecD (67.5 kDa), YidC (63 kDa), SecY
(50 kDa), SecF (35 kDa), SecE (15 kDa), SecG (14 kDa). The
presence of YajC-CBP (14.7 kDa) needs to be confirmed by
Western blotting using an anti-CBP antibody (see Fig. 1c, lane
1). If the Coomassie stained gel is difficult to interpret, the
presence of SecD, YidC, and SecE can be confirmed by Western
blotting using an anti-His antibody. SecD and SecE are
encoded by polycistrons and part of SecYEG and SecDF protein complexes. Therefore, the other complex subunits are
likely to be also present in the membrane.
3.3 Detergent
Solubilization
1. Add DDM to a final concentration of 1.5% w/v to the membranes (see Note 5).
2. Gently agitate the sample at 4
C (in the cold room) for 2 h by
head-over-tail rotation.
3. Centrifuge solubilized membranes for 1 h in a Ti70 rotor at
45,000 rpm (149,000 Â g) at 4
C (see Note 6).
3.4 NiNTA Affinity
Chromatography (All
Steps at 4
C)
1. Equilibrate 5 mL Ni-NTA resin with ten column volumes of
Ni-NTA buffer (see Note 7 for using a prepacked column).
2. Add the Ni-NTA resin to the solubilized membrane supernatant (from step 3 of Subheading 3.3).
3. Gently agitate for 1 h by head-over-tail rotation.
4. Transfer the sample and beads into a gravity flow column.
Collect the flow-through.
5. Wash with at least 20 column volumes of Ni-NTA buffer (see
Note 8, to change the detergent to LMNG at this step).
6. Wash with three to five column volumes of high salt buffer.
7. Wash with at least five column volumes of Ni-NTA buffer.
8. Wash with 20 column volumes of Ni-NTA wash buffer.
9. Verify that there is no A 280nm signal in the wash fraction using a
NanoDrop spectrophotometer before proceeding.
10. Elute with Ni-NTA elution buffer and collect the eluate in
1.5–2 mL tubes.
11. Determine the protein concentration of each fraction by
A 280nm measurements using a NanoDrop spectrophotometer
(see Note 9).
12. Add 0.1 mM PMSF and cOmplete protease inhibitor cocktail
to the protein-containing fractions.
12
Burak V. Kabasakal et al.
(membranes) in ~5 mL HSGM buffer using a Dounce homogenizer. Add cOmplete EDTA-free protease inhibitor cocktail
and 0.5 mM PMSF (see Note 4).
9. Analyze the membrane fraction using a 15% acrylamide gel to
confirm the presence of SecD (67.5 kDa), YidC (63 kDa), SecY
(50 kDa), SecF (35 kDa), SecE (15 kDa), SecG (14 kDa). The
presence of YajC-CBP (14.7 kDa) needs to be confirmed by
Western blotting using an anti-CBP antibody (see Fig. 1c, lane
1). If the Coomassie stained gel is difficult to interpret, the
presence of SecD, YidC, and SecE can be confirmed by Western
blotting using an anti-His antibody. SecD and SecE are
encoded by polycistrons and part of SecYEG and SecDF protein complexes. Therefore, the other complex subunits are
likely to be also present in the membrane.
3.3 Detergent
Solubilization
1. Add DDM to a final concentration of 1.5% w/v to the membranes (see Note 5).
2. Gently agitate the sample at 4
C (in the cold room) for 2 h by
head-over-tail rotation.
3. Centrifuge solubilized membranes for 1 h in a Ti70 rotor at
45,000 rpm (149,000 Â g) at 4
C (see Note 6).
3.4 NiNTA Affinity
Chromatography (All
Steps at 4
C)
1. Equilibrate 5 mL Ni-NTA resin with ten column volumes of
Ni-NTA buffer (see Note 7 for using a prepacked column).
2. Add the Ni-NTA resin to the solubilized membrane supernatant (from step 3 of Subheading 3.3).
3. Gently agitate for 1 h by head-over-tail rotation.
4. Transfer the sample and beads into a gravity flow column.
Collect the flow-through.
5. Wash with at least 20 column volumes of Ni-NTA buffer (see
Note 8, to change the detergent to LMNG at this step).
6. Wash with three to five column volumes of high salt buffer.
7. Wash with at least five column volumes of Ni-NTA buffer.
8. Wash with 20 column volumes of Ni-NTA wash buffer.
9. Verify that there is no A 280nm signal in the wash fraction using a
NanoDrop spectrophotometer before proceeding.
10. Elute with Ni-NTA elution buffer and collect the eluate in
1.5–2 mL tubes.
11. Determine the protein concentration of each fraction by
A 280nm measurements using a NanoDrop spectrophotometer
(see Note 9).
12. Add 0.1 mM PMSF and cOmplete protease inhibitor cocktail
to the protein-containing fractions.
12
Burak V. Kabasakal et al.