Tubulin protein, fluorescent, labeled with HiLyte 647TM (TL670M)

Material
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked HiLyte 647^TM (HiLyte is a trademark of Anaspec Inc, CA) at random surface lysines. An activated ester of HiLyte 647^TM 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 76,000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. HiLyte 647^TM labeled tubulin can be detected using a filter set of 600-630 nm excitation and 660-680 emission. HiLyte 647^TM 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), rhodamine (Cat. # TL590M), X-rhodamine (Cat. # TL620M) 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 TL670M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated HiLyte 670^TM 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: HiLyte 647^TM 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 525-625nm illumination (B).

Biological Activity
The biological activity of HiLyte 647 tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of HiLyte 647 labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.

HiLyte 647^TM labeled tubulin is a new product as of February 2010, there are no citations relating to this product at this time. Other similar products provided by Cytoskeleton have been well cited, some examples are described below:

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.