Cycling Tubulin

1. Thaw tubulin
2. Adjust solution to a final buffer concentration equivalent of glycerol PB
3. Incubate at 37 ^o C for 30 min.
4. Layer tubulin onto cushions of BRB80 containing 60% (v/v) glycerol the cushions should fill half a polycarbonate centrifuge tube and be prewarmed to 37 ^o C
5. Spin at 80,000 rpm for 20 min. at 37 ^o C in the TLA-100.3 rotor
6. Aspirate the supernatant above the cushion
7. Rinse the cushion interface 2 x"s with water
8. Aspirate the cushion
9. Break up pellet in BRB80 pre-chilled to 0 ^o C using a dounce / homogenizer for a large volume or a P1000 pipette tip for a small volume.
   ~undefined Note be sure to avoid creating bubbles during this step !
10. Incubate at 0 ^o C for 15 min. to depolimerize the microtubules
11. Sediment at 80,000 rpm for 10 min. at 4 ^o C in the TLA-100.3 rotor
12. Freeze supernatant in small aliquots using liquid nitrogen and store at -80 ^o C
    Tubulin is active for at least one year.

Buffers :
Glycerol PB:
80 mM K-Pipes
5 mM MgCl2
1 mM EGTA pH 6.8
1 mM GTP
33 % (v/v) glycerol

BRB80: (Brinkley Reassembly Buffer)
80 mM K-Pipes
1 mM MgCl2
1 mM EGTA pH 6.8