Cat. # TL620M
X-rhodamine microtubules formed from X-rhodamine labeled tubulin.
Product Uses Include
- Laser based applications
- Monitoring microtubule dynamcs in living cells
- Speckle microscopy
- Formation of fluorescent microtubules
- Microscopy studies of MAP and microtubule associated motor activities
- Nanotechnology
Material
Bovine brain tubulin (>99% pure, see Cat. # TL238) has been modified to contain covalently linked X-rhodamine at random surface lysines. An activated ester of X-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 66,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. X-rhodamine labeled tubulin can be detected using a filter set of 540-560 nm excitation and 610-630 emission. X-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 488TM (Cat. # TL488M), rhodamine (Cat. # TL590M) and HiLyte 647TM (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 X-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: X-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 green light illumination (B). |
Biological Activity
The biological activity of X-rhodamine tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of X-rhodamine labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
Examples of publications where fluorescent tubulin has been cited
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.
| Product description | Cat. # | Amount | Price & Order |
| Tubulin protein, fluorescent, X-rhodamine labeled, lyophilized | TL620M-A | 5 x 20 µg |
|
| Tubulin protein, fluorescent, X-rhodamine labeled, lyophilized | TL620M-B | 20 x 20 µg |
|
