Method for the Purification of Eukaryotic Ribosomes
Allen Furst
Beckman Coulter, Inc.
Materials
- Buffer A: 0.25M sucrose, 0.05M Tris-HCl (pH 7.5), 1mM dithiothreitol (DTT), 0.1mM EDTA
- Solid KCl
- Sucrose cushion solution: 1.0 M sucrose, 0.5 M KCl, 0.02 M Tris-HCl (pH 7.5), 2mM MgCl2, 0.1mM EDTA
- Buffer B: 0.5M KCl, 0.05 M Hepes-KOH (pH 7.5), 2mM MgCl2, 1mM puromycin, 0.5M KCl
- Buffer C: 0.5M KCl, 0.05M Hepes-KOH (pH 7.5), 5mM MgCl2, 1mM DTT, 0.1mM EDTA
- Subunit storage buffer: 0.25M sucrose, 1mM DTT, 0.1mM EDTA, 10mM KCl, 1mM MgCl2
It is convenient to use polycarbonate centrifuge bottles for this application.
Method
Reticulocyte lysate
Prepared reticulocyte lysates from rabbit may be purchased or prepared by the method of Hershko, et al (1). Briefly, cells from reticulocyte-rich blood (phenylhydrazine-treated animals) are pelleted and washed twice in PBS. Cells are then lysed by addition of 1.5 volumes 1mM dithiothreitol, and centrifuged for 30 min. at 30,000 x g (or equivalent) to remove particulates. A fixed-angle rotor is used for this application for rapid pelleting of materials larger than about 200 S.
The following rotors and centrifugation conditions are suitable for this application.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| JA-25.50 | 8 x 50mL | 16,000 | 0:30 | Avanti J-30I, J-25 Series, J-26 XP Series and J-E |
| Type 70 Ti | 8 x 39mL | 18,000 | 0:30 | Optima L Series |
| MLA-80 | 8 x 8mL | 21,000 | 0:30 | Optima MAX |
Store lysates at -80º C.
Polysomes
Polysomes are prepared from cell lysate by pelleting at 100,000 x g. A fixed-angle rotor is used for this application for rapid pelleting.
- Fill the tubes of an ultracentrifuge rotor with the lysate.
- Centrifuge at 100,000 x g for 3-4 hr. at 4º C.
The following rotors and centrifugation conditions are suitable for this application.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| Type 70 Ti | 8 x 39mL | 34,000 | 3:00 | Optima L Series |
| Type 45 Ti | 6 x 94mL | 32,000 | 3:00 | Optima L Series |
| MLA-80 | 8 x 8mL | 42,000 | 3:00 | Optima MAX |
Ribosomes
Salt-washed ribosomes, without translational factors, are prepared from disrupted polysomes by further centrifugation at 180,000 x g.
- Wash polysome pellet in Buffer A.
- Resuspend the polysome pellet in (2-5mL/pellet) Buffer A. Stir gently on ice.
- Add KCl to 0.5M. Stir 30 min on ice to dissolve.
- Centrifuge the solution for 2 hr at 180,000 x g at 4º C. A fixed angle rotor is used for rapid pelleting.
The following rotors and centrifugation conditions are suitable for this application.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| Type 70 Ti | 8 x 39mL | 50,000 | 2:00 | Optima L Series |
| MLA-80 | 8 x 8mL | 60,000 | 2:00 | Optima MAX |
- Resuspend the pellets in a small amount of Buffer A (0.2-0.5OD units/mL) and store at -80º C or colder.
The supernatant may be used as a source of initiation & elongation factors(2).
Alternatively, ribosomes may be prepared by centrifugation through a sucrose cushion. Polysomes in a storage Buffer A are layered over a sucrose cushion solution in a ratio of 2 parts polysome cushion to 1 part cushion. This preparation is centrifuged for 2-3 hr at 275,000 x g, and the pellets resuspended and stored as described above. The following rotors and centrifugation conditions are suitable for this application. A fixed angle rotor is used for efficient pelleting.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| Type 70.1 Ti | 12 x 13.5mL | 65,000 | 3:00 | Optima L Series |
| MLA-80 | 8 x 8mL | 74,000 | 2:30 | Optima MAX |
| TLA-110 | 8 x 5mL | 83,000 | 2:30 | Optima MAX/TLX |
Ribosomal subunits
Ribosomal subunits may be released from isolated whole 80 S ribosomes by limited exposure to puromycin, a chain terminator which causes the release of nascent polypeptide chains, and then purified on a linear sucrose gradient.
- Dilute the storage solution 10X into Buffer B (puromycin solution), to give an OD260 of about 50 units/mL.
- Incubate 10 min at 0º C.
- Incubate 10 min at 37º C.
- Incubate 5 min at 0º C and layer on a 5-20% sucrose gradient made up in Buffer C.
- The buffer is then centrifuged to separate the 40 S and 60 S subunits. A swinging bucket rotor is used to provide maximum resolution for this application.
The following rotors and centrifugation conditions are suitable for this application. Centrifugation should proceed at 4º C.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| SW 32.1 Ti | 6 x 16.8mL | 32,000 | 4:45 | Optima L Series |
| SW 28.1 Ti | 6 x 16.8mL | 28,000 | 5:45 | Optima L Series |
| MLS-50 | 4 x 5mL | 50,000 | 2:30 | Optima MAX |
- Fractionate gradient and monitor by OD280. Two peaks corresponding to 40 S and 60 S subunits are detected.
- Pool fractions and concentrate by centrifugation. Avoid cross-contamination during the pooling process, although yield may suffer.
The following rotors and centrifugation conditions are suitable for this application. Centrifugation should proceed at 4º C.
| Rotor | Volume | RPM | Time | Applicable Centrifuge |
| Type 70.1 Ti | 12 x 13.5mL | 70,000 | 10:00 | Optima L Series |
| MLA-80 | 8 x 8mL | 74,000 | 7:00 | Optima MAX |
| TLA-110 | 8 x 5mL | 83,000 | 3:30 | Optima MAX/TLX |
Store at -80º C in subunit storage buffer.
References
- Hershko, A.; Heller, H.; Elias, S.; Ciechanover, A. (1983) "Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown." J. Biol. Chem. 258: 8206-8214.
- Merrick, W. C. (1979) "Purification of protein synthesis initiation factors from rabbit reticulocytes." Methods in Enzymology 60: 108-123. Crystal, R. G.; Elson, N. A.; Anderson, W. F. (1974) "Initiation of globin synthesis: Assays." Methods in Enzymology 30: 101-127.
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
