| 3 mg PC-Purified Tubulin (6mg/ml) |
| 10 mM Mg·GTP |
| 100 mM NEM |
| 2-mercaptoethanol (100%=14.3 M) |
| PM Buffer = | 100 mM Pipes pH 6.9 1 mM EGTA 1 mM MgCl2 |
| Sephadex G25 column equilibrated with PM Buffer |
| 1. | Isolate the tubulin from porcine brains as described (Williams & Lee 1982). Modify 3 mg of phosphocellulose-purified tubulin by treating with NEM. |
| 2. | Add to the tubulin Mg·GTP to 0.1 mM and NEM to 1 mM, and incubate the reaction mix for 10 min on ice. |
| 3. | Quench the NEM with 8 mM 2-mercaptoethanol and incubate for an additional 10 min on ice. |
| 4. | Remove excess NEM and 2-mercaptoethanol by passing the reaction mix over a Sephadex G25 column equilibrated with PM buffer. Divide the peak fraction into 50-100 microliter aliquots. Freeze in liquid N2 and store at -80°C. |
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Created 5 April 1999 18:30 GMT
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Modified 9 May 2001 18:55 GMT