DEAE - or phosphocellulose-purified tubulin may be used for derivitizations.
However, I find it faster and more efficient to remove MAPs from cycled
microtubule protein with 0.5 M PIPES by the following procedure:
1) 2X cycled microtubule protein (10-20 mg/ml) in PEM buffer (0.1M PIPES, 1.0mM EGTA, 0.5mM
MgCl2, pH 6.9) is adjusted to O.5M PIPES, 1mM MgCl2, 1mM GTP, 10% DMSO by
addition of an equal volume of 1M PIPES,pH 6.9,1/60 volume 100mM MgCl2, 1/50
volume 100mM GTP,and 1/5 volume DMSO.
2) Incubate 37 C, 10 min - it should become
very turbid and viscous.
3) Pellet MTs at 20,000 x g, 20 min, 37 C (18,000 rpm in an SS-34 rotor).
4) Remove the supernatant which contains the MAPs (although there are
some residual MAPs, they do not survive the conjugation and cycling
procedure).
5) Estimate the pellet volume.
6) Resuspend the pellet in 2-4 volumes
of cold PEM by up and down pipetting with a cut off yellow Eppendorf tip.
7) Incubate at 0 C, 10 min.
8) Add 1mM GTP and 10% DMSO.
9) Incubate 37 C,10 min.
Polymerized MTs are ready for derivitization.
Preparation of Cy3 labeled tubulin.
Cy3 succinimidyl ester (Research Organics) -- Cy3 is sold in
5-packs of vials each containing sufficient dye to label 1 mg of IgG. I have
found 1 vial is sufficient to label 3-4 mg of tubulin or 40-50 microliters of pelleted
MTs.
1) Dissolve 1 vial of Cy3 in 20 microliters of dry DMSO
2) Add to polymerized MTs from 9)
above.
3) Incubate 37 C,10 min. The reaction can be terminated by adding 5mM Potassium
Glutamate, but omission of this step has little effect on labeling stoichiometry.
4) Pellet MTs at 37 C.
5) Resuspend pellet in 5x volume of cold PEM.
6) Incubate 0 C, 10 min.
7) Sediment at 20,000 x g, 20 min, 4 C (In 1.5mL Eppendorf tubes at 18,000 rpm in an SS-34 rotor) to
remove denatured protein and unreacted reagent.
8)Adjust the supernatant to
10% DMSO, 1mM GTP.
9) Incubate 37 C, 10 min to polymerize Mts.
10) Pellet MTs at 37 C.
11) Repeat steps 5)-10) above.
12) Resuspend MT pellet in cold PEM or chosen injection buffer.
13) Incubate 0 C, 10 min.
14) Sediment at 4 C to clarify.
15) Freeze aliquots in liquid nitrogen.
To calculate Dye to Protein Ratio, I use Extinction coefficient of Cy3=130,000 at lambda=554 and determine
protein by BCA or Bradford using BSA as a standard. Dye/Protein Ratio is about
0.5. Yields are typically 30-50%.