Cat. # TL331M, T331M & T331

Product Uses Include

• Monitoring microtubule dynamcs in living cells by microinjection (TL331M & T331M)
• Formation of fluorescent microtubules (TL331M, T331M & T331)
• Microscopy studies of MAP and microtubule associated motor activities (TL331M, T331M & T331)
• Nanotechnology (TL331M, T331M & T331)
  
  

Material
Bovine brain tubulin (>99% pure, see Cat. # TL238) has been modified to contain covalently linked rhodamine at random surface lysines. An activated ester of the fluorochrome [(5-(and 6)-carboxytetramethylrhodamine succinimidyl ester] 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 70,000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. Rhodamine labeled tubulin can be detected using a filter set of 535 nm excitation and 585 emission.

Rhodamine tubulin is available in three formats:

• Lyophilized (Cat. # TL331M) - The most affordable, versatile, stable and easily shipped format. When dissolved in G-PEM, it is identical to T331M. When dissolved in G-PEM with 5% glycerol, it is identical to T331.
• Frozen (Cat. # T331M) - The same product as TL331M but in frozen format
• Frozen containing 5% glycerol (Cat. # T331) - Used for formation of fluorescent microtubules in vitro

Cytoskeleton, Inc. also offers fluorescein labeled tubulin of the same quality (Cat. T332M).

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 TL331M 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 after rhodamine conjugation was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).

Examples of publications where this product was used
Azuma, Y., Arnaoutov, A., Anan, T. and Dasso, M. (2005). PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes. EMBO J.

Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A. and Scholey, J. M. (2004). Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation. Proc. Natl. Acad. Sci. U. S. A. 101, 15938-15943.

Chen, W. and Zhang, D. (2004). Kinetochore fibre dynamics outside the context of the spindle during anaphase. Nat. Cell Biol. 6, 227-231.

Cho, H. P., Liu, Y., Gomez, M., Dunlap, J., Tyers, M. and Wang, Y. (2005). The dual-specificity phosphatase CDC14B bundles and stabilizes microtubules. Mol. Cell. Biol. 25, 4541-4551.

Evans, K. J., Gomes, E. R., Reisenweber, S. M., Gundersen, G. G. and Lauring, B. P. (2005). Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing. J. Cell Biol. 168, 599-606.

Georgi, A. B., Stukenberg, P. T. and Kirschner, M. W. (2002). Timing of events in mitosis. Curr. Biol. 12, 105-114.

Hirst, L. S., Parker, E. R., Abu-Samah, Z., Li, Y., Pynn, R., MacDonald, N. C. and Safinya, C. R. (2005). Microchannel systems in titanium and silicon for structural and mechanical studies of aligned protein self-assemblies. Langmuir 21, 3910-3914.

Laurent, C. E., Delfino, F. J., Cheng, H. Y. and Smithgall, T. E. (2004). The human c-Fes tyrosine kinase binds tubulin and microtubules through separate domains and promotes microtubule assembly. Mol. Cell. Biol. 24, 9351-9358.

Rogers, G. C., Rogers, S. L., Schwimmer, T. A., Ems-McClung, S. C., Walczak, C. E., Vale, R. D., Scholey, J. M. and Sharp, D. J. (2004). Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature 427, 364-370.

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.