

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