6. Resuspend the cell pellet in 3 mL PBS.
7. Centrifuge samples at 4,500 Â g for 20 min at 18–20
C.
8. Aspirate supernatant (at this stage the pellet may be stored at
À80
C for future use, seeNote 2).
3.2 Total Protein
Extraction
1. Resuspend bacterial cell pellets in the 50 μL 100 мМ Tris–HСl
pH 7.6 with 50 μL lysozyme (1 mg/mL).
2. Vortex the suspension at full speed for 2–3 min to disrupt the
bacterial colonies. All clumps should be broken up.
3. Incubate at 4
C for 30 min.
4. Add 100 μL 100 mМ Tris–HСl pH 7.6 (seeNote 3) and 2 μL
of Protease Inhibitor Mix (seeNote 4) to the tube and vortex.
5. Lyse samples on a BeadBeater (MPBio, FastPrep-24) homogenizer with 0.5 mm silica–zirconium beads in the ratio of 1 sample volume to 1 buffer volume. The procedure is repeated two
times for 2 min with a 5-min break on ice.
6. Add 30 μL 20% SDS and 20 μL 1 M DTT.
7. Incubate in a solid thermostat for 30 min at 60
C.
8. Centrifuge in a microfuge at 13,000 Â g and 4
C for 5 min.
9. Recover the upper aqueous phase (approx. 250 μL) (at this
stage the protein extract may be stored at À80
C for future
use).
10. Determine the amount of protein according to Bradford
[14]. Prepare protein dilution standards using the Bradford
Protein Assay Kit. Add the Bradford reagent to the analyzed
samples and standards in a 1:1 ratio (for example 10 μL to
10 μL in a microplate), incubate at room temperature for
10 min. Measure the protein concentration using a Mark spectrophotometer by quantifying the light absorption at a wavelength of 595 nm.
3.3 Gel
Electrophoresis
1. Prepare 1 Â Tris-glycine buffer from the 5 Â stock.
2. For the 7.5% resolving gel (10 mL), mix 4.15 mL deionized
water, 1.9 mL 40% acrylamide, 3.75 mL 1 М Tris–HCl pH 8.8,
100 μL 10% SDS, 8 μL TEMED, and 100 μL 10% PSA and cast
gel in the Mini-Protean glass plates.
3. For the staking gel (2 mL), mix 1.48 mL deionized water,
0.25 mL 40% acrylamide, 0.25 mL 1 М Tris–HCl pH 6.8,
20 μL 10% SDS, 2 μL TEMED, and 20 μL 10% PSA (seeNote
5) and cast gel in the Mini-Protean. Insert a 10-well gel comb
immediately without introducing air bubbles.
4. Take equal amounts of the total protein extract sample, add
2 Â Laemmli sample buffer and heat for 5 min at 95
C.
196
Julia Bespyatykh et al.
7. Centrifuge samples at 4,500 Â g for 20 min at 18–20
C.
8. Aspirate supernatant (at this stage the pellet may be stored at
À80
C for future use, seeNote 2).
3.2 Total Protein
Extraction
1. Resuspend bacterial cell pellets in the 50 μL 100 мМ Tris–HСl
pH 7.6 with 50 μL lysozyme (1 mg/mL).
2. Vortex the suspension at full speed for 2–3 min to disrupt the
bacterial colonies. All clumps should be broken up.
3. Incubate at 4
C for 30 min.
4. Add 100 μL 100 mМ Tris–HСl pH 7.6 (seeNote 3) and 2 μL
of Protease Inhibitor Mix (seeNote 4) to the tube and vortex.
5. Lyse samples on a BeadBeater (MPBio, FastPrep-24) homogenizer with 0.5 mm silica–zirconium beads in the ratio of 1 sample volume to 1 buffer volume. The procedure is repeated two
times for 2 min with a 5-min break on ice.
6. Add 30 μL 20% SDS and 20 μL 1 M DTT.
7. Incubate in a solid thermostat for 30 min at 60
C.
8. Centrifuge in a microfuge at 13,000 Â g and 4
C for 5 min.
9. Recover the upper aqueous phase (approx. 250 μL) (at this
stage the protein extract may be stored at À80
C for future
use).
10. Determine the amount of protein according to Bradford
[14]. Prepare protein dilution standards using the Bradford
Protein Assay Kit. Add the Bradford reagent to the analyzed
samples and standards in a 1:1 ratio (for example 10 μL to
10 μL in a microplate), incubate at room temperature for
10 min. Measure the protein concentration using a Mark spectrophotometer by quantifying the light absorption at a wavelength of 595 nm.
3.3 Gel
Electrophoresis
1. Prepare 1 Â Tris-glycine buffer from the 5 Â stock.
2. For the 7.5% resolving gel (10 mL), mix 4.15 mL deionized
water, 1.9 mL 40% acrylamide, 3.75 mL 1 М Tris–HCl pH 8.8,
100 μL 10% SDS, 8 μL TEMED, and 100 μL 10% PSA and cast
gel in the Mini-Protean glass plates.
3. For the staking gel (2 mL), mix 1.48 mL deionized water,
0.25 mL 40% acrylamide, 0.25 mL 1 М Tris–HCl pH 6.8,
20 μL 10% SDS, 2 μL TEMED, and 20 μL 10% PSA (seeNote
5) and cast gel in the Mini-Protean. Insert a 10-well gel comb
immediately without introducing air bubbles.
4. Take equal amounts of the total protein extract sample, add
2 Â Laemmli sample buffer and heat for 5 min at 95
C.
196
Julia Bespyatykh et al.