protein-enriched fractions from spent culture media, mycobacterial
cells, and several extraction and separation methods that may be
applied to these fractions or extended into clinical samples (such as
tissue homogenates and cell lines). The methods we describe in this
chapter follow the processes we utilize for manipulation of Mycobacterium spp. cells to generate discreet protein fractions and has
been optimized to permit the extraction or separation of major
protein families from a single-cell harvest.
2 Materials
2.1 M. tuberculosis
Inactivation by Gamma
Irradiation
1. M. tuberculosis live cell pellet (see Notes 1 and 2).
2. 70% ethanol.
3. 2.5% Vesphene.
4. JL Shepherd Model 81-14 sealed source Cs-137 Irradiator.
5. Glass tubes, 25 Â 150 mm.
6. Magnetic stir bar, small.
7. 7H9-OADC-Tw medium: Add 4.7 g of Middlebrook 7H9
dehydrated broth to 900 mL of water, stir well. Add 2 mL of
glycerol and allow to disperse fully before adding 2.5 mL of
20% Tween 80 solution. Autoclave at 121
C, 15 psi, for at
least 20 min, then place in a 55
C water bath for 30 min.
Aseptically, add 100 mL of OADC solution (see Note 3).
8. M. tuberculosis cells, actively growing.
9. Disposable inoculation loop, 10μL, sterile.
10. Spectrophotometer.
11. 96-Well plate, sterile, two plates per sample, with lids.
12. Alamar Blue reagent.
13. Microplate reader (see Note 4).
2.2 Concentration
of Culture Filtrates
from Mycobacterium
spp. (See Note 5)
1. Culture filtrate proteins (CFP) from Mycobacterium spp.:
obtained by sterile filtration of spent culture medium after
growth of cells to late-log phase (see Note 6).
2. 90% isopropanol.
3. 70% ethanol.
4. 10 mM ammonium bicarbonate (NH 4 HCO 3 ): 0.79 g/L in
water.
5. Pyrogen-free, 0.2μm filtered water.
6. Amicon stirred ultrafiltration cell (see Note 7).
7. Stainless-steel dispensing pressure vessel or reservoir (Millipore
Sigma).
Extraction and Separation of Mycobacterial Proteins
79
cells, and several extraction and separation methods that may be
applied to these fractions or extended into clinical samples (such as
tissue homogenates and cell lines). The methods we describe in this
chapter follow the processes we utilize for manipulation of Mycobacterium spp. cells to generate discreet protein fractions and has
been optimized to permit the extraction or separation of major
protein families from a single-cell harvest.
2 Materials
2.1 M. tuberculosis
Inactivation by Gamma
Irradiation
1. M. tuberculosis live cell pellet (see Notes 1 and 2).
2. 70% ethanol.
3. 2.5% Vesphene.
4. JL Shepherd Model 81-14 sealed source Cs-137 Irradiator.
5. Glass tubes, 25 Â 150 mm.
6. Magnetic stir bar, small.
7. 7H9-OADC-Tw medium: Add 4.7 g of Middlebrook 7H9
dehydrated broth to 900 mL of water, stir well. Add 2 mL of
glycerol and allow to disperse fully before adding 2.5 mL of
20% Tween 80 solution. Autoclave at 121
C, 15 psi, for at
least 20 min, then place in a 55
C water bath for 30 min.
Aseptically, add 100 mL of OADC solution (see Note 3).
8. M. tuberculosis cells, actively growing.
9. Disposable inoculation loop, 10μL, sterile.
10. Spectrophotometer.
11. 96-Well plate, sterile, two plates per sample, with lids.
12. Alamar Blue reagent.
13. Microplate reader (see Note 4).
2.2 Concentration
of Culture Filtrates
from Mycobacterium
spp. (See Note 5)
1. Culture filtrate proteins (CFP) from Mycobacterium spp.:
obtained by sterile filtration of spent culture medium after
growth of cells to late-log phase (see Note 6).
2. 90% isopropanol.
3. 70% ethanol.
4. 10 mM ammonium bicarbonate (NH 4 HCO 3 ): 0.79 g/L in
water.
5. Pyrogen-free, 0.2μm filtered water.
6. Amicon stirred ultrafiltration cell (see Note 7).
7. Stainless-steel dispensing pressure vessel or reservoir (Millipore
Sigma).
Extraction and Separation of Mycobacterial Proteins
79