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GENERATING FLUORESCENT MICROTUBULES

Materials:
  • Unlabeled tubulin: Cycled Tubulin™ (Cat. No. 032005) or Lyophilized Tubulin (Cat. No. 142001)
  • Labeled tubulin: Alexa Fluor® 488 (Cat. No. 048805), 594 (Cat. No. 059405), or 647 (Cat. No. 064705)
  • 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:
  • Protect from light
  • Adjust the labeled:unlabeled tubulin ratio as needed
  • 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. Combine labeled tubulin at 0.2 mg/ml and unlabeled tubulin at 1.8 mg/ml in 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