7. Transfer the dislodged pellet to a prechilled Wheaton
homogenizer.
8. Wash the emptied ultracentrifuge tube bottom with 500 μL of
ice-cold low-salt HMA buffer and transfer the content in the
homogenizer. Repeat this wash one more time.
9. Homogenize the pellet using the handheld pestle until a
homogeneous suspension is obtained.
10. Transfer the homogenate to a 50 mL centrifuge tube, wash the
homogenizer twice, each time with 1 mL of ice-cold low-salt
HMA buffer. The total volume of the homogenate in the
centrifuge tube at this stage should be 4 mL.
11. Add 6 μL of DNase (TURBO, 2 U/mL) to the homogenate,
and incubate on ice for 1 h.
12. Add 4 mL of ice-cold high-salt HMA buffer to the homogenate, and incubate for further 2 h on ice (see Note 10).
13. Centrifuge the homogenate at 13,000 rpm (22,000 Â g) for
15 min at 4
C to remove large insoluble materials. Use the
centrifuge and rotor as described in step 4.
14. Transfer the supernatant to an ultracentrifuge tube of rotor
similar to TY70Ti, and centrifuge the content in an ultracentrifuge similar to Beckman Coulter Optima L-90K at
42,800 rpm (~188,000 Â g, r max ) for 2 h and 15 min at 4
C.
15. Discard the supernatant, and add 500 μL of ice-cold low-salt
HMA buffer to the pellet. Soak overnight on ice.
16. Transfer the contents to a fresh microfuge tube. Rinse the
ultracentrifuge tube with another 500 μL of ice-cold low-salt
HMA buffer, pool the contents, and centrifuge using a benchtop refrigerated centrifuge similar to Eppendorf 5417R at
13,000 rpm (17,900 Â g) for 15 min at 4
C. Transfer the
supernatant to a fresh microfuge tube, yielding 1 mL of crude
ribosomes containing 30S, 50S, and 70S subunits. Measure the
OD 260 of the crude ribosome. OD 260 of 1.0 is equivalent to
~24 picomoles of ribosomes. Further purification of individual
subunits can be achieved by sucrose gradient fractionation.
3.5 Preparation
of Continuous 10–40%
Sucrose Gradient
1. Place an empty 40 mL polypropylene ultracentrifuge tube for a
rotor similar to Beckman Coulter SW28 in a gradient marker
block. Mark the tube that aligns with the metal rim of the
marker block, which is supplied with the Gradient Station.
2. Draw ~25 mL of 10% sucrose in low-salt HMA buffer using a
syringe, and dispense it in the marked 40 mL ultracentrifuge
tube until it fills up till the marker.
3. Draw ~25 mL of 40% sucrose in low-salt HMA buffer using a
syringe, and gently dispense it in the bottom of the tube until it
fills up till the marker. The 40% sucrose buffer will settle at the
bottom, displacing the 10% sucrose to the top.
Purification of Three Types of Ribosomes from Mycobacteria
159
homogenizer.
8. Wash the emptied ultracentrifuge tube bottom with 500 μL of
ice-cold low-salt HMA buffer and transfer the content in the
homogenizer. Repeat this wash one more time.
9. Homogenize the pellet using the handheld pestle until a
homogeneous suspension is obtained.
10. Transfer the homogenate to a 50 mL centrifuge tube, wash the
homogenizer twice, each time with 1 mL of ice-cold low-salt
HMA buffer. The total volume of the homogenate in the
centrifuge tube at this stage should be 4 mL.
11. Add 6 μL of DNase (TURBO, 2 U/mL) to the homogenate,
and incubate on ice for 1 h.
12. Add 4 mL of ice-cold high-salt HMA buffer to the homogenate, and incubate for further 2 h on ice (see Note 10).
13. Centrifuge the homogenate at 13,000 rpm (22,000 Â g) for
15 min at 4
C to remove large insoluble materials. Use the
centrifuge and rotor as described in step 4.
14. Transfer the supernatant to an ultracentrifuge tube of rotor
similar to TY70Ti, and centrifuge the content in an ultracentrifuge similar to Beckman Coulter Optima L-90K at
42,800 rpm (~188,000 Â g, r max ) for 2 h and 15 min at 4
C.
15. Discard the supernatant, and add 500 μL of ice-cold low-salt
HMA buffer to the pellet. Soak overnight on ice.
16. Transfer the contents to a fresh microfuge tube. Rinse the
ultracentrifuge tube with another 500 μL of ice-cold low-salt
HMA buffer, pool the contents, and centrifuge using a benchtop refrigerated centrifuge similar to Eppendorf 5417R at
13,000 rpm (17,900 Â g) for 15 min at 4
C. Transfer the
supernatant to a fresh microfuge tube, yielding 1 mL of crude
ribosomes containing 30S, 50S, and 70S subunits. Measure the
OD 260 of the crude ribosome. OD 260 of 1.0 is equivalent to
~24 picomoles of ribosomes. Further purification of individual
subunits can be achieved by sucrose gradient fractionation.
3.5 Preparation
of Continuous 10–40%
Sucrose Gradient
1. Place an empty 40 mL polypropylene ultracentrifuge tube for a
rotor similar to Beckman Coulter SW28 in a gradient marker
block. Mark the tube that aligns with the metal rim of the
marker block, which is supplied with the Gradient Station.
2. Draw ~25 mL of 10% sucrose in low-salt HMA buffer using a
syringe, and dispense it in the marked 40 mL ultracentrifuge
tube until it fills up till the marker.
3. Draw ~25 mL of 40% sucrose in low-salt HMA buffer using a
syringe, and gently dispense it in the bottom of the tube until it
fills up till the marker. The 40% sucrose buffer will settle at the
bottom, displacing the 10% sucrose to the top.
Purification of Three Types of Ribosomes from Mycobacteria
159