Making Microtubule Seeds

Prechill glycerol, MgCl 2 , and GTP on ice. Rapidly thaw frozen tubulin, then immediately place on ice. Unpolymerized tubulin is labile! 200 µL aliquot of tubulin (cycled)--in BRB80, stored in -70℃ 100 µL glycerol 10 µL 0.1 M MgCl 2 3 µL 0.1 M GTP (pH adjusted to neutral) or 50 µL aliquot of tubulin (cycled)--in BRB80, stored in -70℃ 25 µL glycerol 2.5 µL 0.1 M MgCl 2 0.75 µL 0.1 M GTP (pH adjusted to neutral) Add other ingredients. Mix gently, but thoroughly. Spin tube briefly. Aliquot tubulin to small (500 µL) tubes that have been prechilled on ice. A standard titration of binding activity requires ~15 µLs. Flash freeze aliquots in liquid nitrogen and store at -70℃. To make polymerized MTs: Place "seeds" at 37℃ for 30 min. Add taxol to 10 µM. I add the required amount of taxol in 1 µL (i.e. for a 12 µL aliquot of MT seeds, add 1 µL of 0.13 mM taxol in DMSO). Mix in gently. Incubate an additional 15 min. at 37℃. Taxol-stabilized MTs are stable at room temp. for several days. NOTE: To dilute MTs, make a cocktail similar to that which the seeds are in: 200 µL BRB80 100 µL glycerol 10 µL 0.1 M MgCl 2 Adjust small aliquots to 10 µM taxol prior to use. Making Long Microtubules 100 µls Tubulin (3-5 mg/ml) 3.3 µls 100 mM MgCl 2 1.3 µls 100 mM GTP 2 µls seeds 45 minutes at 37℃ Add Taxol ~7µls 15 minutes at 37℃ 8/25/95 NEW Protocol for Assembling Microtubules •To a tube containing 10µls of 10mg/ml tubulin add: 10µls G-PEM with 30% Glycerol; 0.75 µl of rhodamine labelled tubulin •Place at 35℃ for 10" •remove to room temp. •Immediately add paclitaxel (taxol) to 10µM final (2µls of a 0.1 mM stock taxol into 20µls) •the microtubules formed by this method have a mean length of ~2µm and can be stable for upto several weeks at room temp. G-PEM buffer : 80mM Pipes 1mM MgCl2 1mM EGTA 1mM GTP pH 6.8 - 7.0 10% Glycerol