4 Notes
1. Heat treatment at 80
C for 25 min will effectively kill the
pathogen. Nevertheless, for added safety, subsequent steps
(up to the sample lysis in a homogenizer BeadBeater step)
should be carried out in a laminar flow cabinet.
2. Pellet may be stored at À80
C for future use, but no more
than 3–5 days.
3. At this step, 100–300 μL 100 mМ Tris–HСl pH 7.6 may be
added.
4. Use Protease Inhibitor Mix at a ratio of 1 μL to 100 μL of the
total sample.
5. Add PSA and TEMED just before pouring each of the gels.
Install the glass in the filling table, consistently fill stacking and
separating gels.
6. We do this manipulation (step 1) on a glass surface. Each part,
cut into 1 Â 1 mm pieces, should be transferred to 2 mL tubes
(we use Eppendorf tubes).
7. For best results, the procedure can be repeated several times by
adding a new solution.
8. Tryptic solution 150 μL on one sample: 15 μL 100% ACN,
37.5 μL 200 mM NH 4 HCO 3 , 3 μL trypsin, and 94.5 μL
deionized water.
9. If the whole volume of the solution (150 μL) is absorbed into
the gel, add an additional 150 μL of the solution but without
trypsin. Solution 150 μL on one sample: 15 μL 100% ACN,
37.5 μL 200 mM NH 4 HCO 3 , and 97.5 μL deionized water.
10. Prepare 50% ACN with 5% formic acid per 300 μL of the
solution: 150 μL acetonitrile, 15 μL formic acid, and 135 μL
deionized water.
11. Prepare 75% ACN with 5% formic acid per 300 μL of the
solution: 225 μL acetonitrile, 15 μL formic acid, and 70 μL
deionized water.
12. Prepare 5% ACN with 0.1% formic acid per 20 μL of the
solution: 1 μL acetonitrile, 0.02 μL formic acid, and
18.98 μL deionized water.
References
1. Ma ˚len H, De Souza GA, Pathak S, Søfteland T,
Wiker HG (2011) Comparison of membrane
proteins of Mycobacterium tuberculosis H37Rv
and H37Ra strains. BMC Microbiol 11:18
2. Schubert OT, Mouritsen J, Ludwig C, Ro ¨st
HL, Rosenberger G, Arthur PK et al (2013)
The Mtb proteome library: a resource of assays
to quantify the complete proteome of Mycobacterium tuberculosis. Cell Host Microbe 13
(5):602–612
3. Xiong Y, Chalmers MJ, Gao FP, Cross TA,
Marshall AG (2005) Identification of
200
Julia Bespyatykh et al.
1. Heat treatment at 80
C for 25 min will effectively kill the
pathogen. Nevertheless, for added safety, subsequent steps
(up to the sample lysis in a homogenizer BeadBeater step)
should be carried out in a laminar flow cabinet.
2. Pellet may be stored at À80
C for future use, but no more
than 3–5 days.
3. At this step, 100–300 μL 100 mМ Tris–HСl pH 7.6 may be
added.
4. Use Protease Inhibitor Mix at a ratio of 1 μL to 100 μL of the
total sample.
5. Add PSA and TEMED just before pouring each of the gels.
Install the glass in the filling table, consistently fill stacking and
separating gels.
6. We do this manipulation (step 1) on a glass surface. Each part,
cut into 1 Â 1 mm pieces, should be transferred to 2 mL tubes
(we use Eppendorf tubes).
7. For best results, the procedure can be repeated several times by
adding a new solution.
8. Tryptic solution 150 μL on one sample: 15 μL 100% ACN,
37.5 μL 200 mM NH 4 HCO 3 , 3 μL trypsin, and 94.5 μL
deionized water.
9. If the whole volume of the solution (150 μL) is absorbed into
the gel, add an additional 150 μL of the solution but without
trypsin. Solution 150 μL on one sample: 15 μL 100% ACN,
37.5 μL 200 mM NH 4 HCO 3 , and 97.5 μL deionized water.
10. Prepare 50% ACN with 5% formic acid per 300 μL of the
solution: 150 μL acetonitrile, 15 μL formic acid, and 135 μL
deionized water.
11. Prepare 75% ACN with 5% formic acid per 300 μL of the
solution: 225 μL acetonitrile, 15 μL formic acid, and 70 μL
deionized water.
12. Prepare 5% ACN with 0.1% formic acid per 20 μL of the
solution: 1 μL acetonitrile, 0.02 μL formic acid, and
18.98 μL deionized water.
References
1. Ma ˚len H, De Souza GA, Pathak S, Søfteland T,
Wiker HG (2011) Comparison of membrane
proteins of Mycobacterium tuberculosis H37Rv
and H37Ra strains. BMC Microbiol 11:18
2. Schubert OT, Mouritsen J, Ludwig C, Ro ¨st
HL, Rosenberger G, Arthur PK et al (2013)
The Mtb proteome library: a resource of assays
to quantify the complete proteome of Mycobacterium tuberculosis. Cell Host Microbe 13
(5):602–612
3. Xiong Y, Chalmers MJ, Gao FP, Cross TA,
Marshall AG (2005) Identification of
200
Julia Bespyatykh et al.