Tubulin protein, fluorescent, labeled with rhodamine (TL590M)

Material
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked rhodamine at random surface lysines. An activated ester of rhodamine was used to label the protein. Labeling stoichiometry was determined by spectroscopic measurement of protein and dye concentrations (dye extinction coefficient when protein bound is 64,000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. rhodamine labeled tubulin can be detected using a filter set of 530-550 nm excitation and 580-600 emission. Rhodamine tubulin is in a versatile, stable and easily shipped format. It is ready for micro-injection or in vitro polymerization. Cytoskeleton, Inc. also offers AMCA (Cat. # TL440M), HiLyte 488^TM (Cat. # TL488M), X-rhodamine (Cat. # TL620M) and HiLyte 647^TM (Cat. # TL670M) labeled tubulins of the same quality.

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 TL590M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated rhodamine 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 (Figure 1 B).

Figure 1: Rhodamine tubulin protein purity determination. A 50 µg sample of unlabeled tubulin protein was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue (A). Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02). 20 µg of the same protein sample was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).

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

Microtubule flux and sliding in mitotic spindles of Drosophila embryos. Brust-Mascher, I. and Scholey, J. M. (2002). Mol. Biol. Cell 13, 3967-3975.
Kinetochore fibre dynamics outside the context of the spindle during anaphase. Chen, W. and Zhang, D. (2004). Nat. Cell Biol. 6, 227-231.
De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation. Kim, H. K., Kang, J. G., Yumura, S., Walsh, C. J., Cho, J. W. and Lee, J. (2005). J. Cell Biol. 169, 719-724.
Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules. Krishnamurthy, G., Cheng, W., Lo, M. C., Aulabaugh, A., Razinkov, V., Ding, W., Loganzo,F., Zask, A. and Ellestad, G. (2003). Biochemistry 42, 13484-13495.
Electrical docking of microtubules for kinesin-driven motility in nanostructures. van den Heuvel, M. G., Butcher, C. T., Lemay, S. G., Diez, S. and Dekker, C. (2005a). Nano Lett. 5, 235-241.
High rectifying efficiencies of microtubule motility on Kinesin-coated gold nanostructures. van den Heuvel, M. G., Butcher, C. T., Smeets, R. M., Diez, S. and Dekker, C. (2005b). Nano Lett. 5, 1117-1122.43
Rapid movement of microtubules in axons. Wang, L. and Brown, A. (2002). Curr. Biol. 12, 1496-1501.