29. Draw the sample into a 1 mL disposable syringe. If available,
set the syringe in an automatic syringe pump set to inject the
diluted sample at a rate of 30μL/min.
30. Visualize the sample, adjusting the screen gain, camera level,
and focus as needed. Consult the instrument user manual for
guidance regarding setup and optimization (see Note 46).
31. Capture video of three technical replicates for a minimum of
30 s, using a constant flow for every replicate. Adjust the
capture threshold for analysis; consult the instrument user
manual for guidance (see Note 47).
3.4 Fracturing
of Whole
Mycobacterium spp.
Cells by French Press
(See Note 48)
1. Thaw γ-irradiated Mycobacterium spp. cells (see Subheading
3.1) at 4
C to reduce proteolysis.
2. Dilute cells in breaking buffer at a concentration of 2 g/mL
based on wet weight of cells. Cell suspension should be quite
viscous.
3. Place cell suspension on ice.
4. Inspect the French pressure cell to ensure that the piston, base
unit, and pressure release valve each have two intact and properly installed O-rings (see Note 49).
5. Use a small amount of glycerol to lubricate French pressure cell
piston and base O-ring.
6. Assemble the pressure cell base by attaching the plug, sample
outlet tube, and pressure release knob (see Note 50) (see
Fig. 5).
7. Place French pressure cell piston into cell unit until reaching
“max fill” mark.
8. Turn unit upside-down and place on stand.
9. Add cell suspension from step 3 to French press cell unit,
leaving enough room to attach base to the unit.
10. Loosen the pressure release knob to allow excess air to escape.
Attach base to unit, then tighten pressure release knob.
11. Keeping a tight hold of both the main cell unit and the base,
turn the complete unit right-side up and place in French press
(see Note 51). Be sure that the unit is flush against the bottom
pegs, and that the piston handle is perpendicular to the
bracing bar.
12. Move the lever to the “High” setting and turn the French
press on.
13. Making small adjustments using the pressure release knob,
keep pressure on cell between 1000 and 1500 while collecting
eluent into a collection bottle (see Note 52).
Extraction and Separation of Mycobacterial Proteins
91
set the syringe in an automatic syringe pump set to inject the
diluted sample at a rate of 30μL/min.
30. Visualize the sample, adjusting the screen gain, camera level,
and focus as needed. Consult the instrument user manual for
guidance regarding setup and optimization (see Note 46).
31. Capture video of three technical replicates for a minimum of
30 s, using a constant flow for every replicate. Adjust the
capture threshold for analysis; consult the instrument user
manual for guidance (see Note 47).
3.4 Fracturing
of Whole
Mycobacterium spp.
Cells by French Press
(See Note 48)
1. Thaw γ-irradiated Mycobacterium spp. cells (see Subheading
3.1) at 4
C to reduce proteolysis.
2. Dilute cells in breaking buffer at a concentration of 2 g/mL
based on wet weight of cells. Cell suspension should be quite
viscous.
3. Place cell suspension on ice.
4. Inspect the French pressure cell to ensure that the piston, base
unit, and pressure release valve each have two intact and properly installed O-rings (see Note 49).
5. Use a small amount of glycerol to lubricate French pressure cell
piston and base O-ring.
6. Assemble the pressure cell base by attaching the plug, sample
outlet tube, and pressure release knob (see Note 50) (see
Fig. 5).
7. Place French pressure cell piston into cell unit until reaching
“max fill” mark.
8. Turn unit upside-down and place on stand.
9. Add cell suspension from step 3 to French press cell unit,
leaving enough room to attach base to the unit.
10. Loosen the pressure release knob to allow excess air to escape.
Attach base to unit, then tighten pressure release knob.
11. Keeping a tight hold of both the main cell unit and the base,
turn the complete unit right-side up and place in French press
(see Note 51). Be sure that the unit is flush against the bottom
pegs, and that the piston handle is perpendicular to the
bracing bar.
12. Move the lever to the “High” setting and turn the French
press on.
13. Making small adjustments using the pressure release knob,
keep pressure on cell between 1000 and 1500 while collecting
eluent into a collection bottle (see Note 52).
Extraction and Separation of Mycobacterial Proteins
91