Cat. # T333

Product Uses Include

• Immobilizing tubulin onto a solid surface via streptavidin.
• Creating tubulin or microtubule affinity matrices with streptavidin coated sepharose beads
• High through-put screening using a proximity assay.
• Microinjection into cells followed by electron microscopy of streptavidin conjugated gold particles to determine the cellular localization of the tubulin.
• Nanotechnology
  
  

Material
Bovine brain tubulin (>99% pure, see Cat. # TL238) has been modified so that random surface lysines contain a covalently linked, long-chain biotin derivative. A long-chain biotin derivative was selected for this procedure because it allows the biotin molecules to be spaced far enough away from the tubulin protein so as not to interfere with its activity, e.g., ligand binding to SPA beads or other streptavidin based reagents. Biotin labeled tubulin is supplied as a lyophilized powder.

Cytoskeleton, Inc. also offers biotinylated cancer cell tubulins (Cat. # H003 & H007)

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, transfered to a nitrocellulose membrane and detected with streptavidin alkaline phosphatase (Fig 1). 10 ng of T333 is readily detectable. No free biotin is detectable in the final product.

Figure 1: Purity determination of biotin tubulin. 10 and 100 ng of T333 was run on a 4-20% SDS-PAGE gel, transferred to a nitrocellulose membrane and biotin labeled material was detected with streptavidin alkaline phosphatase.

Examples of publications where this product was used
Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J. and Liu, J., B. C. (2004). Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins. Nano Lett. 4, 817-821.

Beckers, T., Reissmann, T., Schmidt, M., Burger, A. M., Fiebig, H. H., Vanhoefer, U., Pongratz, H., Hufsky, H., Hockemeyer, J., Frieser, M. et al. (2002). 2-aroylindoles, a novel class of potent, orally active small molecule tubulin inhibitors. Cancer Res. 62, 3113-3119.

Hess, H., Clemmens, J., Brunner, C., Doot, R., Luna, S., Ernst, K. H. and Vogel, V. (2005). Molecular self-assembly of "nanowires"and "nanospools" using active transport. Nano Lett. 5, 629-633.

Kasibhatla, S., Gourdeau, H., Meerovitch, K., Drewe, J., Reddy, S., Qiu, L., Zhang, H., Bergeron, F., Bouffard, D., Yang, Q. et al. (2004). Discovery and mechanism of action of a novel series of apoptosis inducers with potential vascular targeting activity. Mol. Cancer Ther. 3, 1365-1374.

Kuo, C. C., Hsieh, H. P., Pan, W. Y., Chen, C. P., Liou, J. P., Lee, S. J., Chang, Y. L., Chen, L. T., Chen, C. T. and Chang, J. Y. (2004). BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res. 64, 4621-4628.

Prinz, H., Ishii, Y., Hirano, T., Stoiber, T., Camacho Gomez, J. A., Schmidt, P., Dussmann, H., Burger, A. M., Prehn, J. H., Gunther, E. G. et al. (2003). Novel benzylidene-9(10H)-anthracenones as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization. J. Med. Chem. 46, 3382-3394.

Tahir, S. K., Han, E. K., Credo, B., Jae, H. S., Pietenpol, J. A., Scatena, C. D., Wu-Wong, J. R., Frost, D., Sham, H., Rosenberg, S. H. et al. (2001). A-204197, a new tubulin-binding agent with antimitotic activity in tumor cell lines resistant to known microtubule inhibitors. Cancer Res. 61, 5480-5485.

Tahir, S. K., Nukkala, M. A., Zielinski Mozny, N. A., Credo, R. B., Warner, R. B., Li, Q., Woods, K. W., Claiborne, A., Gwaltney, S. L., 2nd, Frost, D. J. et al. (2003). Biological activity of A-289099: an orally active tubulin-binding indolyloxazoline derivative. Mol. Cancer Ther. 2, 227-233.