Koshland Web Site/Methods
Making Microtubule Seeds
Making Microtubule "seeds"
  1. Prechill glycerol, MgCl2, and GTP on ice.
  2. Rapidly thaw frozen tubulin, then immediately place on ice. Unpolymerized tubulin is labile!
    200 µL aliquot of tubulin (cycled)--in BRB80, stored in -70°C
    100 µL glycerol
    10 µL 0.1 M MgCl2
    3 µL 0.1 M GTP (pH adjusted to neutral)
    50 µL aliquot of tubulin (cycled)--in BRB80, stored in -70°C
    25 µL glycerol
    2.5 µL 0.1 M MgCl2
    0.75 µL 0.1 M GTP (pH adjusted to neutral)
  3. Add other ingredients. Mix gently, but thoroughly. Spin tube briefly.
  4. Aliquot tubulin to small (500 µL) tubes that have been prechilled on ice. A standard titration of binding activity requires ~15 µLs.
  5. Flash freeze aliquots in liquid nitrogen and store at -70°C.
  6. To make polymerized MTs:
  7. Place "seeds" at 37°C for 30 min.
  8. 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.
  9. Incubate an additional 15 min. at 37°C.
  10. 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 MgCl2
    Adjust small aliquots to 10 µM taxol prior to use.
  11. Making Long Microtubules
    100 µls Tubulin (3-5 mg/ml)
    3.3 µls 100 mM MgCl2
    1.3 µls 100 mM GTP
    2 µls seeds
  12. 45 minutes at 37°C
  13. Add Taxol ~7µls
  14. 15 minutes at 37°C

    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°C 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
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