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CYCLING 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)
  • Glycerol
  • Glycerol Cushion (60% v/v glycerol in Tubulin PEM Buffer)
  • Liquid Nitrogen
Equipment:
  • Ultracentrifuge and rotor (i.e. Beckman TL-100 Tabletop Ultracentrifuge and TLA-100 rotor)
  • Water bath at 37°C
Technical Notes:
  •  Avoid diluting tubulin beyond its critical concentration
  •  Work in a 37°C water bath when tubulin is polymerized, work in an ice bucket when tubulin is depolymerized
  •  Pre-warm/chill necessary buffers, centrifuges, and rotors as needed
Protocol:
1. Reconstitute Lyophilized Tubulin to 40 mg/ml (see respective protocol for details)


2. Polymerize

a. Add GTP to 1 mM

b. Incubate on ice for 5 minutes

c. Incubate in a 37°C water bath for 2 minutes

d. Add 1/2 volume of pre-warmed glycerol

e. Incubate in a 37°C water bath for 40 minutes


3. Pellet Microtubules

a. Layer polymerized microtubules over a pre-warmed glycerol cushion

*cushion should be roughly twice the volume of the polymerization reaction

b. Spin at 70,000 rpm for 30 minutes at 37°C in a pre-warmed ultracentrifuge and rotor

c. Wash the supernatant/cushion interface three times with pre-warmed Tubulin PEM Buffer

d. Pipet off remaining cushion

e. Gently wash the microtubule pellet with pre-warmed Tubulin PEM Buffer


4. Depolymerize

a. Resuspend the microtubule pellet in a minimal amount of ice-cold Tubulin PEM Buffer

*recommended to use 1/2 volume of original polymerization reaction

b. Incubate on ice for 30 minutes with occasionally mixing by pipet


5. Clarify

a. Spin at 70,000 rpm for 15 minutes at 4°C in a pre-chilled ultracentrifuge and rotor

b. Collect the supernatant on ice

*tubulin can be diluted as desired at this point


6. Store

a. Flash freeze in liquid nitrogen and store at -80°C

*aliquot so as to avoid repeated freeze-thaw cycles