7. Repeat steps 2–5. This pellet is the 50% ammonium sulfate cut.
8. Repeat the calculation from step 6 using a desired ammonium
sulfate 70% and starting ammonium sulfate 50%, then repeat
steps 2–5 to obtain the 70% ammonium sulfate cut (see Note
76).
9. Resuspend each pellet separately in 25–30 mL 10 mM ammonium bicarbonate.
10. Prepare dialysis tubing (see Subheading 3.6, steps 18 and 19)
and add sample.
11. Place in 7 L dialysis buffer #1 (see Subheading 2.11, item 4)
and dialyze at 4
C for 4–16 h.
12. Change to fresh dialysis buffer #1 and dialyze for another
4–16 h.
13. Change to dialysis buffer #2 (containing no DTT, see Subheading 2.11, item 5) and dialyze for 4–16 h.
14. Remove the samples from their dialysis membranes and transfer to clean sample tubes.
15. Quantitate the amount of protein by BCA assay (see Note 11).
Based on the assay results, run 3–4μg of each sample on
SDS-PAGE gel to visualize protein bands.
16. Freeze the sample at À80
C for at least 2 h. Loosen the tube
lid or remove the lid and cover loosely with foil, put the sample
tube in a freeze-dry flask, and place on a freeze-dryer until dry.
4 Notes
1. The procedure outlined here has been optimized for
M. tuberculosis live cell pellets. Inactivation and confirmation
of inactivation in other type of materials (i.e., liquid cultures,
tissue samples, body fluids, etc.) will need additional
optimization.
2. Alternative methods of inactivation can be applied depending
on the downstream analysis. For instance, live cell pellet can be
chemically inactivated with solvents such as 2:1 (chloroform:
methanol v/v) and phenol-based reagents such as Trizol. Heat
inactivation (typically at temperatures !80
C) can also be
performed. Each of these inactivation methods needs to be
optimized and validated according to institutional biosafety
practices.
3. If the medium is not allowed to cool before the addition of
OADC, components of the OADC will break down. It is also
acceptable to allow the medium to cool to RT before the
addition of OADC.
Extraction and Separation of Mycobacterial Proteins
99
8. Repeat the calculation from step 6 using a desired ammonium
sulfate 70% and starting ammonium sulfate 50%, then repeat
steps 2–5 to obtain the 70% ammonium sulfate cut (see Note
76).
9. Resuspend each pellet separately in 25–30 mL 10 mM ammonium bicarbonate.
10. Prepare dialysis tubing (see Subheading 3.6, steps 18 and 19)
and add sample.
11. Place in 7 L dialysis buffer #1 (see Subheading 2.11, item 4)
and dialyze at 4
C for 4–16 h.
12. Change to fresh dialysis buffer #1 and dialyze for another
4–16 h.
13. Change to dialysis buffer #2 (containing no DTT, see Subheading 2.11, item 5) and dialyze for 4–16 h.
14. Remove the samples from their dialysis membranes and transfer to clean sample tubes.
15. Quantitate the amount of protein by BCA assay (see Note 11).
Based on the assay results, run 3–4μg of each sample on
SDS-PAGE gel to visualize protein bands.
16. Freeze the sample at À80
C for at least 2 h. Loosen the tube
lid or remove the lid and cover loosely with foil, put the sample
tube in a freeze-dry flask, and place on a freeze-dryer until dry.
4 Notes
1. The procedure outlined here has been optimized for
M. tuberculosis live cell pellets. Inactivation and confirmation
of inactivation in other type of materials (i.e., liquid cultures,
tissue samples, body fluids, etc.) will need additional
optimization.
2. Alternative methods of inactivation can be applied depending
on the downstream analysis. For instance, live cell pellet can be
chemically inactivated with solvents such as 2:1 (chloroform:
methanol v/v) and phenol-based reagents such as Trizol. Heat
inactivation (typically at temperatures !80
C) can also be
performed. Each of these inactivation methods needs to be
optimized and validated according to institutional biosafety
practices.
3. If the medium is not allowed to cool before the addition of
OADC, components of the OADC will break down. It is also
acceptable to allow the medium to cool to RT before the
addition of OADC.
Extraction and Separation of Mycobacterial Proteins
99