Axoneme-Microtubule Preparation

Axoneme-Microtubule Preparation



      1. Quick thaw tube of axonemes, withdraw 10 microliters and quick freeze remainder.

      2. Add 100 microliters of PM buffer at room temperature.

      3. Spin at 22°C for 10 min at 12,000 rpm in microfuge.

      4. Remove supernatant carefully.

      5. Add 10 micrograms PC-purified tubulin onto the tube wall.

      6. Add 10 micrograms of NEM-modified tubulin onto the tube wall.

      7. Mix drops, stir with yellow tip to resuspend pellet.

      8. Add 0.2 microliter of 50 mM Mg·GTP.

      9. Incubate 2-5 min at 37°C.

      10. Add 3.5 microliters of PM + 40 micromolar taxol.

      11. Dilute 1:5 or 1:3 with PM + taxol to give a good density.

    Modified from T. Salmon Laboratory

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Created 7 April 1999 19:00 GMT

Modified 27 March 2002 19:00 GMT