

POLYMERIZING CYCLED TUBULIN™
Materials:
- Cycled Tubulin™ (Cat. No. 032005)
- Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8)
- DTT
- GTP or GMPCPP
- Taxol (optional)
Equipment:
- Water bath at 37°C
Technical Notes:
- Avoid diluting tubulin beyond its critical concentration
- If stabilized microtubules are desired, either polymerize with GMPCPP or stabilize with Taxol
- Add Taxol stepwise to avoid precipitation
- Do not place polymerized microtubules on ice
Protocol:
1. Assemble the Polymerization Reaction
a. Dilute Cycled Tubulin™ to 2 mg/ml with Tubulin PEM Buffer
b. Add DTT and guanosine (GTP or GMPCPP) to 1 mM each
c. Incubate on ice for 5 minutes
2. Polymerize
a. Incubate in a 37°C water bath for 1 hour
3. Stabilize with Taxol (optional)
a. Add 1/10 volume Taxol at 2 uM, incubate in a 37°C water bath for 10 minutes
b. Add 1/10 volume Taxol at 20 uM, incubate in a 37°C water bath for 10 minutes
c. Add 1/10 volume Taxol at 200 uM, incubate in a 37°C water bath for 15 minutes
*include Taxol to 200 uM in subsequent buffers