4. Ultracentrifuge rotor similar to SW 28 rotor (Beckman Coulter) with corresponding 40 mL polypropylene tubes.
5. Ultracentrifuge rotor similar to TY70Ti (Beckman Coulter)
with corresponding 23.6 mL polycarbonate tubes with cap
assemblies.
6. 500 mL 60% sucrose in DEPC-Water: Add 300 g of sucrose in
250 mL DEPC-treated water. Stir the solution until complete
solubilization. Bring the final volume with DEPC-treated
water to 500 mL. Filter-sterilize the solution and store at
room temperature.
7. Density gradient fractionation system (Brandel Inc.).
8. UV spectrophotometer.
3 Methods
3.1 Cell Culture
for Purification of C+,
CÀ, and Mpy-Bound
CÀ Ribosomes from
M. smegmatis
This protocol details cell culture conditions for purifying active CÀ
ribosomes. Specific adjustments are noted for cell culture conditions for purifying hibernating (Mpy-bound) CÀ ribosomes or C+
ribosomes from M. smegmatis.
1. Inoculate 20 mL of 7H9ADCTw with a frozen stock of
M. smegmatis, mc
2
155, and incubate at 37
C on a shaker
until OD 600 reaches 0.8–1.0.
2. Harvest the cells by centrifugation at 4000 rpm (3500 Â g) for
10 min using Thermo Sorvall Legend XTR or similar centrifuge and its swing-out rotor. Resuspend the cells in 20 mL of
PBSTw and centrifuge at 3500 Â g for 10 min at room temperature. Repeat the washing step two additional times, before
resuspending it in 5 mL PBSTw (see Note 2).
3. Inoculate 500 mL of low-zinc Sauton’s medium with washed
M. smegmatis cells from step 2 to a final OD 600 of 0.08–0.1.
Typically, 1:60 dilution of cells in the fresh medium yields this
initial density (see Note 3).
4. Incubate cultures on a platform shaker at 37
C for 28 h,
harvest the cells by centrifugation at 8000 rpm (10,000 Â
g) for 20 min using Thermo Sorvall Lynx 4000 or similar
centrifuge with F14-6 x 250y or similar rotor. Cells can be
frozen at À80
C.
5. For purifying C+ ribosomes, steps 1–4 should be followed as
described with the exception that low-zinc Sauton’s medium
should be replaced with high-zinc Sauton’s medium.
6. For purifying hibernating CÀ ribosomes, steps 1–4 should be
followed as described with the exception that incubation time
for cells should be extended from 28 to 96 h (see Notes 4 and
5 as well as Fig. 1).
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Yunlong Li et al.
5. Ultracentrifuge rotor similar to TY70Ti (Beckman Coulter)
with corresponding 23.6 mL polycarbonate tubes with cap
assemblies.
6. 500 mL 60% sucrose in DEPC-Water: Add 300 g of sucrose in
250 mL DEPC-treated water. Stir the solution until complete
solubilization. Bring the final volume with DEPC-treated
water to 500 mL. Filter-sterilize the solution and store at
room temperature.
7. Density gradient fractionation system (Brandel Inc.).
8. UV spectrophotometer.
3 Methods
3.1 Cell Culture
for Purification of C+,
CÀ, and Mpy-Bound
CÀ Ribosomes from
M. smegmatis
This protocol details cell culture conditions for purifying active CÀ
ribosomes. Specific adjustments are noted for cell culture conditions for purifying hibernating (Mpy-bound) CÀ ribosomes or C+
ribosomes from M. smegmatis.
1. Inoculate 20 mL of 7H9ADCTw with a frozen stock of
M. smegmatis, mc
2
155, and incubate at 37
C on a shaker
until OD 600 reaches 0.8–1.0.
2. Harvest the cells by centrifugation at 4000 rpm (3500 Â g) for
10 min using Thermo Sorvall Legend XTR or similar centrifuge and its swing-out rotor. Resuspend the cells in 20 mL of
PBSTw and centrifuge at 3500 Â g for 10 min at room temperature. Repeat the washing step two additional times, before
resuspending it in 5 mL PBSTw (see Note 2).
3. Inoculate 500 mL of low-zinc Sauton’s medium with washed
M. smegmatis cells from step 2 to a final OD 600 of 0.08–0.1.
Typically, 1:60 dilution of cells in the fresh medium yields this
initial density (see Note 3).
4. Incubate cultures on a platform shaker at 37
C for 28 h,
harvest the cells by centrifugation at 8000 rpm (10,000 Â
g) for 20 min using Thermo Sorvall Lynx 4000 or similar
centrifuge with F14-6 x 250y or similar rotor. Cells can be
frozen at À80
C.
5. For purifying C+ ribosomes, steps 1–4 should be followed as
described with the exception that low-zinc Sauton’s medium
should be replaced with high-zinc Sauton’s medium.
6. For purifying hibernating CÀ ribosomes, steps 1–4 should be
followed as described with the exception that incubation time
for cells should be extended from 28 to 96 h (see Notes 4 and
5 as well as Fig. 1).
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Yunlong Li et al.