Axoneme-Microtubule Preparation Copyright 1996-2003. All rights reserved.
Materials
10 microliters Axonemes
PM Buffer =
100 mM PIPES pH 6.9
1 mM EGTA
1 mM MgCl2
50 mM Mg·GTP
PC-Purified Tubulin (5 mg/mL)
NEM-Modified Tubulin (5 mg/mL)
4 mM Taxol (MW=853.9) in DMSO
Procedure
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
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