Axoneme Preparation

Axoneme Preparation


      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.

    Modified from Allen and Borisy 1974

    H Song and SA Endow


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Created 31 March 1999 19:40 GMT

Modified 06 June 2002 18:55 GMT