Making de-tyrosinated Microtubule for TIRF by VASHI plus SVBP

'''Part I: Got the polymerized MTs (Wild type).

Just use normal protocol we used for microtubule polymerization.'''

'''Part II: remove Tyrosine in C-tail of α-tubulin.

Microtubules in BRB80 (pH 6.8)*:     60 µl

DTT:                                                   1 mM

PMSF:                                                1 mM

VASH1: SVBP:                                   10 nM

*: Since we resuspend polymerized microtubules in 60 µl of BRB80 plus 20 µM Taxol, and the concentration of microtubules probably be 1mg/ml, ~10 µM.

1.     The concentration of VASH1: SVBP mixture of our stock is 22 µM, dilute it 1 to 50 with GF150 buffer (or other buffers pH close to 7), Adding 1.5 µl of dilution into 60 µl of polymerized microtubules, plus 1 mM of DTT and 1 mM of PMSF. Incubate 4 to 24 hours at ℃ in water bath.

2.     Adding 6 µl of DTT into reaction above (final concentration of DTT is 10mM) to terminate the reaction.

3.     Spin over 150 µl of sucrose cushion (25% sucrose) with Taxol in BRB80 (20 µM Taxol), 100 000x g, 10 minutes at ℃.

4.     Resuspend pellet with 60ul of BRB80-Taxol (20 µM Taxol), adding 0.6 µl of GTP and 0.6 µl of Gentamicin, keep at room temperature.

5.     Checking the de-tyrosinated microtubules on TIRF (1 to 100 dilution first). For 647 labeled tubulin, sometimes there are some debris, in this case, just take some of polymerized microtubules, spin over 150 µl of 25% sucrose (in BRB80 with 20uM taxol) on the normal centrifuge (full speed), resuspend the pellet and checking with TIRF again after dilution. (This is an optional step)