"Materials"

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MT ring

Preparation of biotin labeled tubulin

1. Tubulin was thawed, and polymerized at 37 0C for 15 min.
2. Biotin-XX-SE (Molecular Probes, Cat. B-1606) was dissolved at 0.1 M in dry dimethyl sulfoxide (DMSO).
3. Biotin-XX-SE solution was added to tubulin, while pipetting to distribute it rapidly to a final concentration of 2 mM. incubate at 37 0C for 20 min.
4. Mixture was layered onto cushions and spun at 54,000 rpm for 1h at 37 0C.
5. Pellets were resuspended and spun cold, being careful to wash the cushion inter face well to remove all the biotin-XX-SE.
6. Tubulin was depolymerized and spun 40,000 rpm for 15 min at 4 0C.
7. Steps (3) – (6) were performed to give once cycled biotin- tubulin.
8. Steps (3) – (6) were repeated to give twice cycled biotin- tubulin. The final pellet is resuspended in BRB80.
9. The final biotin-tubulin is frozen and stored as per the cycled tubulin.

Determination of stoichiometry
Tubulin concentration was determined by SDS-PAGE electrophores.
To quantify biotin, we use defference of avidin and avidin-biotin complex in
spectroscopic characteristic because avidin combines stoichiometrically with biotin.
The dye 4-hydroxyazobenzene-2’-carboxylic acid (HABA), which binds only to avidin with changing spectrum,
so that it can be used as an indicator for unoccupied binding sites [1].

1. Prepare standard curve.
Biotin standard solutions [(0, 10, 20, 50, 100, 200, 300, 500 µM) biotin,
80 mM PIPES, 5 mM MgCl2, 1 mM EGTA] were prepared.
Each biotin standard solutions was added to avidin solution of which final concentration is
0.4 mg mL-1 avidin, 250 mM HABA, 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA.
The mixtures were left for 15 min at roomtemperature.
A500 of these solutions were measured to prepare standard curve.

2. Determination of biotin.
1　mg mL-1 Pronase was added to biotin-labeled tubulin, and left for 1 h at 37 0C.
This mixture was added to avidin solution, and left for 15 min at roomtemperature.
A500 of this solution were measured to determine concentration of biotin.

Mechanism of constructing ring shaped microtubules

Surface-adhered kinesin motor proteins can transport biotinylated

microtubules.
With adding streptavidin (St), partially St coated microtubules were collide

and collisions of the moving microtubules were enables to cross-linking
when gliding microtubules were encountered.

biotin-streptavidin

Biotin, a 244 dalton compound, is bound with exceptionally high affinity (Ka =

2.5x1013 M)
by the 53 kDa protein Streptavidin, due to the extremely slow unbinding rate of

the bond.