5. Centrifuge at 10,000 Â g for 5 min at room temperature,
transfer the supernatant into a new tube.
6. Load samples along with 3 μL of the PageRuler Prestained
Protein Ladder to the 7.5% SDS-gel and run gel for
60–80 min at 120 V in running buffer using the Bio-Rad
Mini Protean system.
7. Disassemble the Bio-Rad Mini Protean glass plates and stain
the gel with the Coomassie G-250 for 1–2 h. Rinse the gel with
10% acetic acid solution.
3.4 In-Gel Trypsin
Digestion
1. Cut out the right part of the gel (a band containing proteins),
divided into six parts, cut each one into 1 Â 1 mm pieces by
using the carbon steel sterilized scalpels (seeNote 6).
2. Wash the gels from Coomassie G-250 with 50% ACN in
50 mM NH 4 HCO 3 solution. Remove the solution after discoloration of the gels (seeNote 7).
3. Cover up the gels with 10 mM DTT in 100 mM NH 4 HCO 3
solution, incubate for 30 min at 56
C. Remove the solution.
4. Wash the gels with 55 mM IAA in 100 mM NH 4 HCO 3
solution, incubate for 20 min at room temperature in the
dark. Remove the solution.
5. Dry washed gels by adding 100% acetonitrile, incubate for
20 min at room temperature, remove acetonitrile, leave tubes
open until the gels are completely dry.
6. Add a solution of trypsin (20 ng/μL) in 40 mM NH 4 HCO 3
and 10% acetonitrile (seeNote 8) to the dried gels, incubate 1 h
on ice (seeNote 9) and then not less than 16 h at 37
C.
7. Add a 5% formic acid (2 volume, 300 μL) to the sample and
incubate at room temperature for 30 min. Transfer the solution
to a separate tube.
8. Add a 50% ACN with 5% formic acid solution (seeNote 10) to
the sample and incubate at room temperature for 30 min.
Transfer the solution to a separate tube.
9. Add a 75% ACN with 5% formic acid solution (seeNote 11) to
the sample and incubate at room temperature for 30 min.
Transfer the solution to a separate tube.
10. The extracts are combined, dried completely by vacuum drying
(SpeedVac), and dissolve in a minimum volume (approx.
20 μL) of 5% ACN with and 0.1% formic acid solution (seeNote
12).
Proteogenomic Analysis of Mycobacteria
197
transfer the supernatant into a new tube.
6. Load samples along with 3 μL of the PageRuler Prestained
Protein Ladder to the 7.5% SDS-gel and run gel for
60–80 min at 120 V in running buffer using the Bio-Rad
Mini Protean system.
7. Disassemble the Bio-Rad Mini Protean glass plates and stain
the gel with the Coomassie G-250 for 1–2 h. Rinse the gel with
10% acetic acid solution.
3.4 In-Gel Trypsin
Digestion
1. Cut out the right part of the gel (a band containing proteins),
divided into six parts, cut each one into 1 Â 1 mm pieces by
using the carbon steel sterilized scalpels (seeNote 6).
2. Wash the gels from Coomassie G-250 with 50% ACN in
50 mM NH 4 HCO 3 solution. Remove the solution after discoloration of the gels (seeNote 7).
3. Cover up the gels with 10 mM DTT in 100 mM NH 4 HCO 3
solution, incubate for 30 min at 56
C. Remove the solution.
4. Wash the gels with 55 mM IAA in 100 mM NH 4 HCO 3
solution, incubate for 20 min at room temperature in the
dark. Remove the solution.
5. Dry washed gels by adding 100% acetonitrile, incubate for
20 min at room temperature, remove acetonitrile, leave tubes
open until the gels are completely dry.
6. Add a solution of trypsin (20 ng/μL) in 40 mM NH 4 HCO 3
and 10% acetonitrile (seeNote 8) to the dried gels, incubate 1 h
on ice (seeNote 9) and then not less than 16 h at 37
C.
7. Add a 5% formic acid (2 volume, 300 μL) to the sample and
incubate at room temperature for 30 min. Transfer the solution
to a separate tube.
8. Add a 50% ACN with 5% formic acid solution (seeNote 10) to
the sample and incubate at room temperature for 30 min.
Transfer the solution to a separate tube.
9. Add a 75% ACN with 5% formic acid solution (seeNote 11) to
the sample and incubate at room temperature for 30 min.
Transfer the solution to a separate tube.
10. The extracts are combined, dried completely by vacuum drying
(SpeedVac), and dissolve in a minimum volume (approx.
20 μL) of 5% ACN with and 0.1% formic acid solution (seeNote
12).
Proteogenomic Analysis of Mycobacteria
197