[39] Preparation of modified tubulins

Publisher Summary This chapter presents a collection of the various different ways by which tubulins are modified to generate probes for investigating microtubule (MT) dynamics in vitro and in vivo . Labeling with biotin and various fluorochromes is described, as well as the preparation of N-ethylmaleimide tubulin, which has been used to block minus-end growth in vitro . The use of GTP analogs to prepare stable labeled microtubules has proved very useful in a number of different experiments. The tubulin used in the presented methods was prepared from bovine brain by two cycles of temperature-dependent polymerization, followed by phosphocellulose chromatography. The cycling procedure described in the chapter selects active subunits and removes free nucleotide. This produces a tubulin preparation suitable for use in in vitro assays. The standard biotin-labeled tubulin preparation has been used to determine sites of microtubule elongation in vivo and in vitro . It is difficult to quantitate the stoichiometry of biotin labeling on a routine basis, but early work using radioactive N-hydroxysuccinimide (NHS)-biotin gave a labeling stochiometry of one to three biotins/tubulin dimer. The final yield of twice cycled biotin-tubulin is about 10% of the starting protein. Tetramethylrhodamine-labeled tubulin has been used to follow microtubules in living cells and it is also used for marking microtubules in real-time in vitro assays.