13. Analyze the input, flow-through, wash, and eluate fractions by
SDS PAGE using a 15% acrylamide gel (see Fig. 1c for a typical
result).
14. Pool the protein-containing fractions.
3.5 Desalting Step
(at 4
C)
1. Equilibrate a 5 mL desalting column with CBP-binding buffer
using the AKTA system (see Notes 10 and 11).
2. Inject 3 mL of Ni-NTA eluate (use combined proteincontaining fractions from step 14 of Subheading 3.4) onto
the desalting column (see Note 12).
3. Collect 1 mL fractions and pool protein-containing fractions.
Repeat this procedure until all holo-translocon-containing eluate fractions from step 14 of Subheading 3.4 are in
CBP-binding buffer.
3.6 Calmodulin
Affinity
Chromatography
(at 4
C)
1. Equilibrate 5 mL calmodulin affinity resin with CBP-binding
buffer.
2. Add the pooled fractions from step 3 of Subheading 3.5 to the
resin.
3. Incubate overnight at 4
C and gently agitate.
4. Transfer the sample into a gravity flow column.
5. Wash the column with at least ten column volumes of CBP
washing buffer.
6. Verify by NanoDrop measurements that there is no A 280nm
signal in the last wash fraction before proceeding to the
next step.
7. Elute with five column volumes of CBP elution buffer and
collect fractions.
8. Determine the concentration of each fraction by NanoDrop
A 280nm measurements (see Note 9).
9. Analyze the input, flow-through, wash, and eluate fractions by
SDS PAGE using a 15% acrylamide gel.
10. Pool holo-translocon-containing fractions.
3.7 Sample
Concentration
1. Equilibrate a 100 kDa molecular weight cut-off concentrator
with S6 Buffer.
2. Load the holo-translocon-containing fractions from step 10 of
Subheading 3.6 into the concentrator.
3. Centrifuge at 3500 Â g, 4
C until the sample volume is
reduced to 500 μL (monitor volume regularly).
Production of Multi-subunit Membrane Protein Complexes
13
SDS PAGE using a 15% acrylamide gel (see Fig. 1c for a typical
result).
14. Pool the protein-containing fractions.
3.5 Desalting Step
(at 4
C)
1. Equilibrate a 5 mL desalting column with CBP-binding buffer
using the AKTA system (see Notes 10 and 11).
2. Inject 3 mL of Ni-NTA eluate (use combined proteincontaining fractions from step 14 of Subheading 3.4) onto
the desalting column (see Note 12).
3. Collect 1 mL fractions and pool protein-containing fractions.
Repeat this procedure until all holo-translocon-containing eluate fractions from step 14 of Subheading 3.4 are in
CBP-binding buffer.
3.6 Calmodulin
Affinity
Chromatography
(at 4
C)
1. Equilibrate 5 mL calmodulin affinity resin with CBP-binding
buffer.
2. Add the pooled fractions from step 3 of Subheading 3.5 to the
resin.
3. Incubate overnight at 4
C and gently agitate.
4. Transfer the sample into a gravity flow column.
5. Wash the column with at least ten column volumes of CBP
washing buffer.
6. Verify by NanoDrop measurements that there is no A 280nm
signal in the last wash fraction before proceeding to the
next step.
7. Elute with five column volumes of CBP elution buffer and
collect fractions.
8. Determine the concentration of each fraction by NanoDrop
A 280nm measurements (see Note 9).
9. Analyze the input, flow-through, wash, and eluate fractions by
SDS PAGE using a 15% acrylamide gel.
10. Pool holo-translocon-containing fractions.
3.7 Sample
Concentration
1. Equilibrate a 100 kDa molecular weight cut-off concentrator
with S6 Buffer.
2. Load the holo-translocon-containing fractions from step 10 of
Subheading 3.6 into the concentrator.
3. Centrifuge at 3500 Â g, 4
C until the sample volume is
reduced to 500 μL (monitor volume regularly).
Production of Multi-subunit Membrane Protein Complexes
13