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