Thaw tubulin Adjust solution to a final buffer concentration equivalent of glycerol PB Incubate at 37 o C 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 37 o C Spin at 80,000 rpm for 20 min. at 37 o C 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 0 o C 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 ! Incubate at 0 o C for 15 min. to depolimerize the microtubules Sediment at 80,000 rpm for 10 min. at 4 o C in the TLA-100.3 rotor 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
