LEVEL III
Additional analysis of the interaction of tubulin and MAPs can be made following the separation of the proteins. This can be accomplished by phophocellulose chromatography, as described in Aamodt and Williams. 9 Once separated, the polymerization can be monitored with varying ratios of tubulin to MAP, and in the presence of various polymerization inhibitors (colchicine, vincristine).
Other studies could be made of isolated cellular structures, such as flagella, cilia, or even isolated spindle fibers. The latter can be isolated in quantity from sea urchin embryos, as outlined by K.A. Suprenant. 10
Collect egg and sperm by injection of KCL, and combine the
two for fertilization. As the embryo's reach metaphase (80-85
minutes at 18° C), they are collected, washed free
of salt water and homogenized in a low pH PIPES buffer containing
Mg
, EGTA and Triton X-100 detergent. The spindles are pelleted
at 1000 Xg (clinical centrifuge), washed several times and
monitored with polarization microscopy. Since the spindles are
highly birefringent, they can be studied without the use of a
TEM. They can be observed fairly readily with standard enhanced
phase contrast optics or stained for bright field optics.
The sea urchin also serves as a ready source of flagella (sperm) and cilia (developing pluteus). Virtually all of the early work on tubulin polymerization was accomplished with this model system.
Cell Biology Laboratory Manual