storage solution from the resin. Allow 20 min for the wash to
drain from the column. Replace the cap after draining is
complete.
12. Gently transfer the 10 mg of diluted 100 kDa retentate from
step 9 to the column (see Note 44); take care not to cause
excess disruption of the resin bed. Allow the sample and resin
to settle for 5 min.
13. Place a clean collection tube under the gravity flow column,
remove the cap, and allow the sample to flow through the resin.
14. Cap the gravity flow column and apply the collected material to
the resin again to remove any remaining material below
700 kDa. Allow the sample and resin to settle for 5 min.
15. Place a new collection tube under the gravity flow column,
remove the cap, and allow the sample to flow through the resin.
16. Leaving the cap open, add 1 mL of PBS and collect the wash in
the tube from step 15.
17. Repeat step 16. The total final volume will be roughly 7 mL.
18. Rinse a 3 kDa centrifugal filter with PBS prior to use, as
described in step 2.
19. Add the sample from step 17 to the 3 kDa centrifugal filter.
Spin the filter at its maximum centrifugal force at 4
C until the
sample has reduced to the desired volume.
20. Transfer the retentate to a new tube.
21. Rinse the filter membrane with 200μL PBS and add the rinse to
the retentate sample. The eluate can be discarded.
22. Filter the vesicle sample using a leur-lock syringe and 0.22μm
PES syringe filter into a new, sterile tube. Keep the sample at
4
C for short-term storage.
23. Quantify the protein content of the sample by MicroBCA assay,
following the manufacturer’s protocol.
24. Take 5μg each of the original CFP sample (see step 1), the
100 kDa eluate (see step 8), the 100 kDa retentate (see step 8),
and the purified vesicle sample (see step 22) for SDS-PAGE gel.
25. Visualize the proteins by silver staining or Coomassie
® blue
staining following standard recommendations to evaluate soluble protein reduction (Fig. 3).
26. Optional: Western blotting may be performed to confirm the
presence or absence of specific proteins (see Note 45).
27. Optional: TEM can also be performed to visualize size and
vesicle integrity (Fig. 4).
28. Dilute 5μg of the vesicle sample (as quantified in step 23) to
1 mL using PBS; vortex for 15 s on medium speed.
Extraction and Separation of Mycobacterial Proteins
89
drain from the column. Replace the cap after draining is
complete.
12. Gently transfer the 10 mg of diluted 100 kDa retentate from
step 9 to the column (see Note 44); take care not to cause
excess disruption of the resin bed. Allow the sample and resin
to settle for 5 min.
13. Place a clean collection tube under the gravity flow column,
remove the cap, and allow the sample to flow through the resin.
14. Cap the gravity flow column and apply the collected material to
the resin again to remove any remaining material below
700 kDa. Allow the sample and resin to settle for 5 min.
15. Place a new collection tube under the gravity flow column,
remove the cap, and allow the sample to flow through the resin.
16. Leaving the cap open, add 1 mL of PBS and collect the wash in
the tube from step 15.
17. Repeat step 16. The total final volume will be roughly 7 mL.
18. Rinse a 3 kDa centrifugal filter with PBS prior to use, as
described in step 2.
19. Add the sample from step 17 to the 3 kDa centrifugal filter.
Spin the filter at its maximum centrifugal force at 4
C until the
sample has reduced to the desired volume.
20. Transfer the retentate to a new tube.
21. Rinse the filter membrane with 200μL PBS and add the rinse to
the retentate sample. The eluate can be discarded.
22. Filter the vesicle sample using a leur-lock syringe and 0.22μm
PES syringe filter into a new, sterile tube. Keep the sample at
4
C for short-term storage.
23. Quantify the protein content of the sample by MicroBCA assay,
following the manufacturer’s protocol.
24. Take 5μg each of the original CFP sample (see step 1), the
100 kDa eluate (see step 8), the 100 kDa retentate (see step 8),
and the purified vesicle sample (see step 22) for SDS-PAGE gel.
25. Visualize the proteins by silver staining or Coomassie
® blue
staining following standard recommendations to evaluate soluble protein reduction (Fig. 3).
26. Optional: Western blotting may be performed to confirm the
presence or absence of specific proteins (see Note 45).
27. Optional: TEM can also be performed to visualize size and
vesicle integrity (Fig. 4).
28. Dilute 5μg of the vesicle sample (as quantified in step 23) to
1 mL using PBS; vortex for 15 s on medium speed.
Extraction and Separation of Mycobacterial Proteins
89