Making Microtubule Seeds
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- 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
