Fluorescein tubulin (T332M)

Material
Bovine brain tubulin (>99% pure, see Cat. # TL238) has been modified to contain covalently linked fluorescein at random surface lysines. An activated ester of the fluorochrome was used to label the protein. Labeling stoichiometry was determined by spectroscopic measurement of protein and dye concentrations. Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. Fluorescein labeled tubulin can be detected using a filter set of 488 nm excitation and 535 emission.

Cytoskeleton, Inc. also offers rhodamine labeled tubulin of the same quality (Cat. # TL331M, T331M and T331).

Purity
The protein purity of the tubulin used for labeling is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. The protein used for T332M is >99% pure tubulin. Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated fluorescein dye would be visible in the dye front. No fluorescence is detected in the dye front, indicating that no free dye is present in the final product.

Biological Activity
The biological activity of T332M is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of fluorescein labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.

Examples of publications where this product was used
Cao, T. T., Chang, W., Masters, S. E. and Mooseker, M. S. (2004). Myosin-Va binds to and mechanochemically couples microtubules to actin filaments. Mol. Biol. Cell 15, 151-161.

Ligon, L. A., Shelly, S. S., Tokito, M. and Holzbaur, E. L. (2003). The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization. Mol. Biol. Cell 14, 1405-1417.
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Murray, J. W., Bananis, E. and Wolkoff, A. W. (2000). Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process. Mol. Biol. Cell 11, 419-433.

Schaefer, A. W., Kabir, N. and Forscher, P. (2002). Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones. J. Cell Biol. 158, 139-152.

Tsukada, M., Prokscha, A., Ungewickell, E. and Eichele, G. (2005). Doublecortin association with actin filaments is regulated by neurabin II. J. Biol. Chem. 280, 11361-11368.