| 600 mL Chlamydomonas reinhardtii ida 1 mt+ (strain CC-2664) |
| 10 mM HEPES pH 7.2 |
| HMDS Buffer = | 10 mM HEPES pH 7.2 5 mM MgCl2 1 mM DTT 4% (wt/vol) Sucrose |
| Nonidet P-40 (NP40) |
| 0.5 M EGTA pH 7 |
| HMDS-25 Buffer = HMDS + 25% (wt/vol) Sucrose |
| Salt Extraction Buffer = | 10 mM HEPES pH 7.2 0.6 M NaCl 5 mM MgCl2 1 mM EGTA 7 mM 2-mercaptoethanol 1 mM DTT |
| Axoneme Storage Buffer = | 10 mM HEPES pH 7.2 0.5 mM EGTA 1 mM MgCl2 3.5 mM 2-mercaptoethanol 1 mM DTT 1 mM Mg-GTP 50% (wt/vol) glycerol |
| 1. | Culture Chlamydomonas for three days under a fluorescent light. Harvest cells by centrifuging at 230 x g for 7 min at 22°C. Wash cells in 150 mL distilled water and pellet. Resuspend resulting pellet in 150 mL of 10 mM Hepes pH 7.2 and pellet again. |
| 2. | Resuspend cells in 10 mL of cold HMDS and deflagellate by adding Nonidet P-40 to 0.02%. Mix gently and invert on ice for 4 min. |
| 3. | Add an equal volume of ice cold HMDS + 0.5 mM EGTA and remove cell bodies by centrifuging at 1600 x g for 10 min at 4°C in a Sorvall SS-34 Rotor. |
| 4. | Gently layer the resulting supernatant onto a 5 mL cushion of HMDS-25 and then centrifuge at 20,000 x g for 30 min at 4°C. |
| 5. | Resuspend the pellet of flagellae in 1 mL of salt extraction buffer and incubate for 30 min at 22°C with occasional swirling to remove axonemal membranes and dyneins. |
| 6. | Centrifuge the extraction mix in a microfuge at 12,000 rpm for 15 min at room temperature. Resuspend the resulting pellet in 300 microliters of axoneme storage buffer and store in 20 microliter aliquots at -80°C after freezing in liquid N2. |
| 1. | The use of Chlamydomonas dynein mutants helps prevent leaching of dyneins from the axonemes during motility assays. The C. reinhardtii ida 1 mutant is defective in the inner dynein arms. The mutant strain can be obtained from the Chlamydomonas Stock center at Duke University. |
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Created 31 March 1999 19:40 GMT
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Modified 06 June 2002 18:55 GMT