|PC - Purifed tubulin (alpha/beta tubulin MW = 100,000)|
| PM = 100 mM |
| PIPES, pH 6.9 |
|4 mM Taxol (MW = 853.9) in DMSO|
|50 mM Mg-GTP =|| 50 µl 100 mM GTP, pH ~7 |
50 µl 100 mM Mg2SO4
|1.|| Thaw frozen tubulin by swirling in 37°C waterbath. |
Adjust concentration to 40 µM with PM.
Add 50 mM Mg-GTP to 1 mM.
Incubate 30 minutes at 37°C.
|2.|| Warm equal volume of 40 µM taxol diluted in PM to 37°C. |
Add in 2-3 aliquots to tubulin + GTP, mix gently.
Incubate 5 minutes at 37°C.
|3.|| Aliquot MTs and flash freeze in liquid nitrogen.|
Final = 2 mg/ml (20 µM) tubulin/MTs
0.5 mM Mg-GTP
20 µM taxol
1 X PM buffer
Modified from T. Salmon Laboratory
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Created 2 March 1999 21:00 GMT
Modified 27 March 2002 18:55 GMT