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At relatively high centripetal forces, polymerized microtubules will sediment while unpolymerized tubulin protein will remain suspended in solution. This biophysical property can be leveraged in probing for microtubule-associated proteins and assessing tubulin polymerization competency (when stabilizing factors such as taxol are excluded). This protocol outlines how to fractionate tubulin protein solutions by centrifugation and probe the resulting pellet and supernatant fractions.
Assemble the polymerization mix on ice.
Incubate the polymerization mix on ice for 5 minutes. This allows the tubulin protein to bind GTP.
Clarify the polymerization mix to remove any protein aggregates. Spin at 90,000 rpm (350,000 x g) for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100). Collect the supernatant on ice in a clean microtube.
Induce polymerization by incubating the polymerization mix in a 37°C water bath for 1 hour.
Add taxol to the polymerized microtubules in the following stepwise manner. Continue incubating in a 37°C water bath:
Mix the protein of interest with the taxol-stabilized microtubules at 1 μM each in 100 μl reaction buffer (10 mM HEPES, pH=7.7, 50 mM KCl, 1 mM DTT, 20 μM taxol).
Incubate the reaction at room temperature for 15 minutes.
Layer the reaction over a 150 ul sucrose cushion (10 mM HEPES, pH 7.7, 50 mM KCl, 20 μM taxol, 40% sucrose w/v) in an ultracentrifuge rotor (i.e. TLA 100). Spin at 60,000 rpm (150,000 x g) for 20 minutes at 26°C.
Analyze the pellet and supernatant fractions by SDS-PAGE. Polymerization reactions devoid of GTP can serve as a negative control.