Koshland Web Site/Methods
Cycling Tubulin
  1. Thaw tubulin
  2. Adjust solution to a final buffer concentration equivalent of glycerol PB
  3. Incubate at 37oC 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 37oC
  5. Spin at 80,000 rpm for 20 min. at 37oC 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 0oC using a dounce / homogenizer for a large volume or a P1000 pipette tip for a small volume.
    * Note be sure to avoid creating bubbles during this step !
  10. Incubate at 0oC for 15 min. to depolimerize the microtubules
  11. Sediment at 80,000 rpm for 10 min. at 4oC in the TLA-100.3 rotor
  12. Freeze supernatant in small aliquots using liquid nitrogen and store at -80oC
    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

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