3.8 Size Exclusion
Chromatography
(Optional)
1. Equilibrate a Superose 6 column with water and then with two
column volumes of S6 Buffer (see Note 13).
2. Load 500 μL of sample from step 3 of Subheading 3.7 onto the
column.
3. Collect 0.5 mL fractions.
4. Analyze the protein-containing fractions on a 15% SDS
PAGE gel.
Pool the peak fractions (Fig. 1d), if required concentrate to
~1 mg/mL using a concentrator with 100 kDa molecular weight
cut-off. Freeze aliquots in liquid nitrogen and store the purified
holo-translocon at À80
C.
4 Notes
1. For each membrane protein complex, the growth temperature
and the optimal time point to harvest the cells have to be
determined experimentally. It is recommended to also test
different strains (Fig. 1b).
2. It is possible to freeze the cells in liquid nitrogen and store the
cells at À80
C at this step.
3. Alternatively, two passages through a microfluidizer or cell
disrupter at 25 kPsi can be used to open the cells.
4. It is possible to freeze the dissolved membranes in liquid nitrogen and store at À80
C after this step.
5. For each new membrane protein complex, detergent screens
have to be tested to identify a suitable mild, non-denaturing,
solubilizing detergent [26], ideally followed by an activity assay
(Fig. 1b). Different screens have been reported [27, 28], various commercial detergent screens are available (e.g., Qiagen,
Jena Bioscience).
6. If the centrifuge tube is not completely filled, it is recommended to either fill the Ti70 centrifugation tubes to the top
with buffer or to reduce the centrifugation speed.
7. Alternatively, use a HisTrap FF column (GE Healthcare) coupled to a peristaltic pump for loading and washing the sample.
Prior to loading, the supernatant from ultracentrifugation is
passed through a 0.22 μM filter to further remove any large
particles. During the washing step, the color of the column will
change from brown to almost light blue over the course of
about 1 h. During this time, detergent exchange (to LMNG for
example) or supplementation of any additive (e.g., cholesteryl
hemisuccinate) can be performed. For elution, the column is
connected to an AKTA chromatography system.
14
Burak V. Kabasakal et al.
Chromatography
(Optional)
1. Equilibrate a Superose 6 column with water and then with two
column volumes of S6 Buffer (see Note 13).
2. Load 500 μL of sample from step 3 of Subheading 3.7 onto the
column.
3. Collect 0.5 mL fractions.
4. Analyze the protein-containing fractions on a 15% SDS
PAGE gel.
Pool the peak fractions (Fig. 1d), if required concentrate to
~1 mg/mL using a concentrator with 100 kDa molecular weight
cut-off. Freeze aliquots in liquid nitrogen and store the purified
holo-translocon at À80
C.
4 Notes
1. For each membrane protein complex, the growth temperature
and the optimal time point to harvest the cells have to be
determined experimentally. It is recommended to also test
different strains (Fig. 1b).
2. It is possible to freeze the cells in liquid nitrogen and store the
cells at À80
C at this step.
3. Alternatively, two passages through a microfluidizer or cell
disrupter at 25 kPsi can be used to open the cells.
4. It is possible to freeze the dissolved membranes in liquid nitrogen and store at À80
C after this step.
5. For each new membrane protein complex, detergent screens
have to be tested to identify a suitable mild, non-denaturing,
solubilizing detergent [26], ideally followed by an activity assay
(Fig. 1b). Different screens have been reported [27, 28], various commercial detergent screens are available (e.g., Qiagen,
Jena Bioscience).
6. If the centrifuge tube is not completely filled, it is recommended to either fill the Ti70 centrifugation tubes to the top
with buffer or to reduce the centrifugation speed.
7. Alternatively, use a HisTrap FF column (GE Healthcare) coupled to a peristaltic pump for loading and washing the sample.
Prior to loading, the supernatant from ultracentrifugation is
passed through a 0.22 μM filter to further remove any large
particles. During the washing step, the color of the column will
change from brown to almost light blue over the course of
about 1 h. During this time, detergent exchange (to LMNG for
example) or supplementation of any additive (e.g., cholesteryl
hemisuccinate) can be performed. For elution, the column is
connected to an AKTA chromatography system.
14
Burak V. Kabasakal et al.