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 rhodamine labeled tubulin.
X-Rhodamine or Tetramethylrhodamine succinimidyl esters obtained from Molecular Probes.
1) Prepare a 10 mg/ml stock solution of Rhodamine-NHS in anhydrous DMSO (the stock can be stored at
-80 C and freeze-thawed; it will last indefinitely if kept dry).
2) Add Rhodamine-NHS stock (add reagent while vortexing to allow thorough mixing) to polymerized
tubulin from 9) to yield a molar ratio of 20 to 1, assuming the tubulin concentration in the MT
pellet measured in 5) is 80 mg/ml. (e.g. 1 mg X-Rhodamine-NHS per 0.1 ml MT pellet is approximately
20:1, since 0.1 ml * 80 mg/ml= 8 mg tubulin and MW(Tubulin)/ MW(X-Rhodamine-NHS) =100,000/632=158;
therefore [1 mg X-Rhodamine-NHS/8 mg Tubulin] * 158 = 20.
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(X-Rhodamine)= 70,000 at
Lamda=584 and Extinction coefficient(TMR)=50,000 at Lamda=556 and determine protein by BCA or
Bradford using a BSA standard. For X-Rhodamine tubulin D/P= 0.5-0.8. For TMR tubulin D/P=0.8-1.2.
Yields are typically 30-50%. Increasing the initial reaction stoichiometry will increase
the D/P, but significantly lower yields. Yields are typically 30-50%.