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POLYMERIZING LYOPHILIZED TUBULIN

Materials:
  • Lyophilized Tubulin (Cat. No. 142001)
  • Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, 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. Reconstitute Lyophilized Tubulin (see respective protocol for details)


2. Assemble the Polymerization Reaction

a. Dilute Lyophilized 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


3. Polymerize

a. Incubate in a 37°C water bath for 1 hour


4. 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