17. French pressure cell (SLM-AMINCO) 40,000 PSI was used in
development of this protocol. If another cell is used, scale
pressures accordingly.
18. Bead beating protocol is suitable for breaking live cells. If
breaking live M. tuberculosis, all procedures must be performed
within a BSL-3 laboratory, following all appropriate biosafety
protocols. Resulting lysate should be inactivated by gamma
irradiation or filtered by passing the lysate through a 0.8μm,
13 mm syringe filter, followed by a repeat filtration through a
0.2μm, 13 mm syringe filter into a sterile cryovial.
19. Beads made of material other than silica can also be used. It is
recommended that beating conditions be optimized for each
type of beads. Bulk beads can also be purchased and used to fill
2.0 mL tubes. If this is chosen, bead volume should not exceed
one-sixth of the tube capacity.
20. These extraction methods were optimized for the extraction of
100 mg of freeze-dried protein sample in 50 mL Oakridge
tubes. Adjust volumes and materials accordingly for smaller
protein extracts. Recovery of extracted proteins is variable
among protein sample types and detergent used for processing.
Average recoveries from 100 mg of total protein sample range
between 40% and 60%, as determined by protein assay.
21. If the biphase does not appear after several h on the first
incubation, or if the biphase is too small to remove, double
the volume with PBS and proceed to the next stirring and
incubation step. A biphase should definitely appear following
the second and third incubations. If it does not, increase the
incubation temperature to 55
C for a short period (approximately 20 min), then return to 37
C until the biphase clarifies.
22. Ethanol may also be used if acetone is unavailable.
23. Use caution while working with phenol. Always handle phenol
in a chemical fume hood, wearing proper PPE, use only glass
pipettes, and ensure chemical compatibility with all tubes and
containers.
24. The live cell pellet should be frozen completely, preferably
overnight, at À80
C before removal from the BSL-3. It is
crucial to follow the training and guidelines of the researcher’s
specific institution when handling the contained live cell pellet
outside of the BSL-3 laboratory.
25. Cells should be transported on ice to avoid thawing and preserve the materials for downstream analysis. Transport should
be approved by institutional biosafety committee and follow
federal and local regulations for the transport of dangerous
goods.
Extraction and Separation of Mycobacterial Proteins
101
development of this protocol. If another cell is used, scale
pressures accordingly.
18. Bead beating protocol is suitable for breaking live cells. If
breaking live M. tuberculosis, all procedures must be performed
within a BSL-3 laboratory, following all appropriate biosafety
protocols. Resulting lysate should be inactivated by gamma
irradiation or filtered by passing the lysate through a 0.8μm,
13 mm syringe filter, followed by a repeat filtration through a
0.2μm, 13 mm syringe filter into a sterile cryovial.
19. Beads made of material other than silica can also be used. It is
recommended that beating conditions be optimized for each
type of beads. Bulk beads can also be purchased and used to fill
2.0 mL tubes. If this is chosen, bead volume should not exceed
one-sixth of the tube capacity.
20. These extraction methods were optimized for the extraction of
100 mg of freeze-dried protein sample in 50 mL Oakridge
tubes. Adjust volumes and materials accordingly for smaller
protein extracts. Recovery of extracted proteins is variable
among protein sample types and detergent used for processing.
Average recoveries from 100 mg of total protein sample range
between 40% and 60%, as determined by protein assay.
21. If the biphase does not appear after several h on the first
incubation, or if the biphase is too small to remove, double
the volume with PBS and proceed to the next stirring and
incubation step. A biphase should definitely appear following
the second and third incubations. If it does not, increase the
incubation temperature to 55
C for a short period (approximately 20 min), then return to 37
C until the biphase clarifies.
22. Ethanol may also be used if acetone is unavailable.
23. Use caution while working with phenol. Always handle phenol
in a chemical fume hood, wearing proper PPE, use only glass
pipettes, and ensure chemical compatibility with all tubes and
containers.
24. The live cell pellet should be frozen completely, preferably
overnight, at À80
C before removal from the BSL-3. It is
crucial to follow the training and guidelines of the researcher’s
specific institution when handling the contained live cell pellet
outside of the BSL-3 laboratory.
25. Cells should be transported on ice to avoid thawing and preserve the materials for downstream analysis. Transport should
be approved by institutional biosafety committee and follow
federal and local regulations for the transport of dangerous
goods.
Extraction and Separation of Mycobacterial Proteins
101