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)
   or
   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
    
    
    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°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