8. 70% ethanol.
9. Acid-fast staining kit.
10. Glycerol.
11. French press (SLM AMINCO).
12. French pressure cell, 40 K (see Note 17).
13. French pressure cell stand.
2.5 Fracturing
of Whole
Mycobacterium spp.
Cells by Bead Beating
1. M. tuberculosis whole cells, inactivated or live (see Note 18).
2. Lysing Matrix B Pre-Filled Silica, 0.1 mm 2.0 mL tubes (see
Note 19).
3. Mini-Bead Beater.
4. 0.8μm/0.22μm syringe filters.
2.6 Subcellular
Fractionation
of Mycobacterium spp.
Whole-Cell
Lysate (WCL)
1. Whole-cell lysate (see Subheading 3.4 or 3.5).
2. Breaking buffer (see Subheading 2.4, item 7).
3. PBS-EDTA (see Subheading 2.4, item 3).
4. Breaking buffer without DNAse or RNAse: 50 mL PBS-EDTA
with protease inhibitor tablet.
5. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
6. 40 mL ultracentrifuge tubes.
7. High-speed centrifuge and rotor.
8. Ultracentrifuge and rotor.
9. Dialysis tubing, 3.5 kDa MWCO.
10. 10 mM ammonium bicarbonate (see Subheading 2.2, item 4).
11. Dialysis tank (7 L).
12. Dialysis clips and foam float.
13. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
14. SDS-PAGE gel.
2.7 Extraction
of Proteins from
Subcellular Fractions
Using SDS (See Note
20)
1. Soluble/insoluble protein sample (such as cell wall or membrane obtained in Subheading 3.6).
2. Phosphate-buffered saline (PBS) (see Subheading 2.3, item 4).
3. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
4. Pyrogen-free, 0.2μm filtered water.
5. High-speed centrifuge and rotors.
6. Micro stir bars.
7. Acetone.
Extraction and Separation of Mycobacterial Proteins
81