Cycling Tubulin
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Thaw tubulin
Adjust solution to a final buffer concentration equivalent of glycerol PB
Incubate at 37oC for 30 min.
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
Spin at 80,000 rpm for 20 min. at 37oC in the TLA-100.3 rotor
Aspirate the supernatant above the cushion
Rinse the cushion interface 2 x"s with water
Aspirate the cushion
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.
~undefined Note be sure to avoid creating bubbles during this step !
Incubate at 0oC for 15 min. to depolimerize the microtubules
Sediment at 80,000 rpm for 10 min. at 4oC in the TLA-100.3 rotor
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
