Tubulin protein, fluorescent, AMCA labeled (TL440M)

Material
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked 7-amino-4-methyl coumarin-3-acetic acid (AMCA) at random surface lysines. An activated ester of X-AMCA 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 40,000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. AMCA labeled tubulin can be detected using a filter set of 350-370 nm excitation and 430-450 emission. AMCA tubulin is in a versatile, stable and easily shipped format. It is ready for micro-injection or in vitro polymerization. Cytoskeleton, Inc. also offers HiLyte 488 (Cat. # TL488M), rhodamine (Cat. # TL590M), X-rhodamine (Cat. # TL620M) and HiLyte 647 (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 TL440M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated AMCA 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: AMCA 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 AMCA tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of AMCA labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.

AMCA 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.