10. Tips 1,000–5,000 μL.
11. рH meter.
12. Tris–HCl pH 7.6.
13. Triton X-100.
14. Phosphate-salt buffer (PBS) made with deionized water.
15. Milli Q water.
16. Wash buffer 1.5 M Tris–HCl, pH 7.5–8. Add 20 mL of water
to a 100 mL graduated cylinder or a glass beaker. Weigh 9.1 g
Tris–HCl and transfer it to the cylinder/beaker. Add 1 mL of
Triton X-100. Mix and adjust pH with HCl. Adjust volume to
50 mL with water. Store at 4
C.
2.2 Total Protein
Extraction
1. 100 mM Tris–HCl pH 7.6 made with deionized water.
2. Lysozyme 1 mg/mL, made with deionized water.
3. Protease inhibitor mix.
4. Milli Q water.
5. Dithiothreitol (DTT).
6. Sodium dodecyl sulfate (SDS), 10% made with deionized
water.
7. 0.5 mm silica–zirconium beads.
8. Bradford Protein Assay Kit.
2.3 Gel
Electrophoresis
1. Tris base.
2. Hydrochloric acid (HCl).
3. Milli Q water.
4. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Add about
100 mL water to a 1 L graduated cylinder or a glass beaker.
Weigh 181.7 g Tris base and transfer it to the cylinder/beaker.
Add water to a volume of 900 mL. Mix and adjust pH with 6 M
HCl. Make up to 1 L with water. Store at 4
C.
5. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 60.6 g
Tris base and prepare a 1 L solution as in the previous step.
Store at 4
C.
6. Ammonium persulfate (PSA): 10% solution in deionized (Milli
Q) water.
7. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED): Store at
4
C.
8. 30% acrylamide/bis-acrylamide solution (29.2:0.8).
9. Dithiothreitol.
Proteogenomic Analysis of Mycobacteria
193