18. Transfer the sample into a 0.2μm PES filter unit and filtersterilize. For maximum recovery, rinse the ultrafiltration disc
with 5 mL 10 mM ammonium bicarbonate and combine with
the concentrated sample prior to filtration.
19. Clean tubing and all stirred ultrafiltration cell parts per manufacturer’s instructions.
20. Quantitate the amount of protein by BCA assay. Based on the
assay results, run 5μg of each sample on an SDS-PAGE gel to
visualize protein bands.
21. Optional: Western blot may be performed to confirm the
presence or absence of specific proteins (see Note 41).
22. Store the sample at À80
C.
3.3 Enrichment
of Mycobacterium spp.
Extracellular Vesicles
1. Thaw CFP (see Subheading 3.2) in a 4
C refrigerator overnight
(see Note 42).
2. Rinse a 100 kDa centrifugal filter with PBS: add PBS to the
unit capacity. Spin the device at its maximum centrifugal force
for 5 min and discard any remaining retentate as well as the
eluate.
3. Fill the 100 kDa centrifugal filter with thawed CFP from step
1. Spin the device at its maximum centrifugal force at 4
C until
the sample has reduced to the desired volume. Retain the eluate
for downstream analysis (see Note 43).
4. Perform buffer exchange: Add PBS to the sample in the
100 kDa centrifugal filter and centrifuge as in step 3 until the
sample has reduced to the desired volume. Save the eluate in a
clean tube and repeat the buffer exchange with addition of PBS
at least three times, pooling the eluate from each buffer
exchange cycle.
5. Recover the retentate in a clean tube.
6. Gently rinse the 100 kDa centrifugal filter membrane with
PBS, and add this to the saved retentate.
7. Quantitate proteins in the eluate and retentate using the BCA
kit and determine protein concentration (see Note 11).
8. Store the eluate at 4
C for confirmation of protein reduction if
desired. If the retentate is not used immediately, store at 4
C.
9. Dilute 10 mg of 100 kDa retentate from step 6 to a volume of
5 mL with PBS, mixing gently.
10. Add 1 mL of well-mixed Capto™ Core 700 multimodal chromatography resin to an empty, capped gravity flow column.
Place the column in a size appropriate rack fitted with a
drip tray.
11. Uncap the bottom of the column and let the liquid drain for
5 min. Once drained, add 10 mL of PBS to wash out the
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Megan Lucas et al.
with 5 mL 10 mM ammonium bicarbonate and combine with
the concentrated sample prior to filtration.
19. Clean tubing and all stirred ultrafiltration cell parts per manufacturer’s instructions.
20. Quantitate the amount of protein by BCA assay. Based on the
assay results, run 5μg of each sample on an SDS-PAGE gel to
visualize protein bands.
21. Optional: Western blot may be performed to confirm the
presence or absence of specific proteins (see Note 41).
22. Store the sample at À80
C.
3.3 Enrichment
of Mycobacterium spp.
Extracellular Vesicles
1. Thaw CFP (see Subheading 3.2) in a 4
C refrigerator overnight
(see Note 42).
2. Rinse a 100 kDa centrifugal filter with PBS: add PBS to the
unit capacity. Spin the device at its maximum centrifugal force
for 5 min and discard any remaining retentate as well as the
eluate.
3. Fill the 100 kDa centrifugal filter with thawed CFP from step
1. Spin the device at its maximum centrifugal force at 4
C until
the sample has reduced to the desired volume. Retain the eluate
for downstream analysis (see Note 43).
4. Perform buffer exchange: Add PBS to the sample in the
100 kDa centrifugal filter and centrifuge as in step 3 until the
sample has reduced to the desired volume. Save the eluate in a
clean tube and repeat the buffer exchange with addition of PBS
at least three times, pooling the eluate from each buffer
exchange cycle.
5. Recover the retentate in a clean tube.
6. Gently rinse the 100 kDa centrifugal filter membrane with
PBS, and add this to the saved retentate.
7. Quantitate proteins in the eluate and retentate using the BCA
kit and determine protein concentration (see Note 11).
8. Store the eluate at 4
C for confirmation of protein reduction if
desired. If the retentate is not used immediately, store at 4
C.
9. Dilute 10 mg of 100 kDa retentate from step 6 to a volume of
5 mL with PBS, mixing gently.
10. Add 1 mL of well-mixed Capto™ Core 700 multimodal chromatography resin to an empty, capped gravity flow column.
Place the column in a size appropriate rack fitted with a
drip tray.
11. Uncap the bottom of the column and let the liquid drain for
5 min. Once drained, add 10 mL of PBS to wash out the
88
Megan Lucas et al.