Cat. # TL238

Product Uses Include

• IC50 & EC50 determinations for anti-tubulin ligands.
• Microtubule binding studies
• Tubulin monomer binding studies
• HDAC6 studies
• Microtubule activated kinesin ATPase assays
  
  

Material
Tubulin protein has been purified from bovine brain by an adaptation of the method of Shelanski et al. (1), Further purification to >99% purity was achieved by cation exchange chromatography. Tubulin is supplied as a white lyophilized powder.

Fully active for polymerization, this product is lyophilized with a patented tecnhnology for increased stability and longevity. TL238 is stable for 1 year at 4°C desiccated. If your project requires the same batch of tubulin for consistent results, it is highly recommended that the item is purchased in bulk in order to save time and money. The protein is identical to T238 when resuspended to 10 mg/ml in G-PEM buffer, and identical to T237 when resuspended to 10 mg/ml in G-PEM plus 10% glycerol. >99% pure tubulin is also available in a convenient lyophilized pre-formed microtubule format (Cat. # MT001) for use in e.g. kinesin ATPase assays and microtubule binding studies.

The porcine (pig brain source) equivalent protein is also available, see Cat.# T240.

Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >99% pure

Figure 1: A 50 µg sample of TL238 protein was separated by electrophoresis on a 4-20% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (cat. # ADV02).

Biological Activity
The biological activity of TL238 is assessed by a tubulin polymerization assay. The ability of tubulin to polymerize into microtubules ise followed by observing an increase in optical density of a tubulin solution at OD340 nm (see Figure 2). Under the experimental conditions defined below a 5 mg/ml tubulin solution in General Tubulin Buffer (Cat. # BST01) buffer plus 5% glycerol (Cat. # BST05) and 1 mM GTP (Cat. # BST06) should achieve an OD340 nm absorption reading between 0.75 - 1.10 in 30 min at 37°C. The assay volume is 180 ul and assumes a spectrophotometer pathlength of 0.8 cm.

It should be noted that tubulin minus glycerol WILL NOT polymerize in G-PEM buffer until very high tubulin concentrations (>10 mg/ml). Even at these concentrations polymerization is comparatively slow. Efficient polymerization at lower concentration of tubulin minus glycerol can be achieved by addition of a polymerization stimulating compound, e.g., glycerol, paclitaxel or DMSO.

Figure 2: Tubulin Polymerization Assay. Polymerization reactions in triplicate contains 180 µl of 5 mg/ml TL238 in 80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCl2, 5% glycerol and 1 mM GTP in a 1/2 area 96-well plate. Polymerization was started by incubation at 37°C and followed by optical density readings at 340 nm. The increase in optical density indicates that >95% of the tubulin is polymerized.

Examples of publications where this product was used
Fan, S., Hurd, T. W., Liu, C. J., Straight, S. W., Weimbs, T., Hurd, E. A., Domino, S. E. and Margolis, B. (2004). Polarity proteins control ciliogenesis via kinesin motor interactions. Curr. Biol. 14, 1451-1461.

Groisman, I., Huang, Y. S., Mendez, R., Cao, Q., Theurkauf, W. and Richter, J. D. (2000). CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell 103, 435-447.

Haggarty, S. J., Koeller, K. M., Wong, J. C., Grozinger, C. M. and Schreiber, S. L. (2003). Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl. Acad. Sci. U. S. A. 100, 4389-4394.

Kar, S., Fan, J., Smith, M. J., Goedert, M. and Amos, L. A. (2003). Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. EMBO J. 22, 70-77.

Larsson, N., Segerman, B., Gradin, H. M., Wandzioch, E., Cassimeris, L. and Gullberg, M. (1999). Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association. Mol. Cell. Biol. 19, 2242-2250.

Moores, C. A., Perderiset, M., Francis, F., Chelly, J., Houdusse, A. and Milligan, R. A. (2004). Mechanism of microtubule stabilization by doublecortin. Mol. Cell 14, 833-839.

Nogales, E., Wolf, S. G. and Downing, K. H. (1998). Structure of the αβ tubulin dimer by electron crystallography. Nature 391, 199-203.

Ovechkina, Y., Wagenbach, M. and Wordeman, L. (2002). K-loop insertion restores microtubule depolymerizing activity of a "neckless" MCAK mutant. J. Cell Biol. 159, 557-562.

Skiniotis, G., Cochran, J. C., Muller, J., Mandelkow, E., Gilbert, S. P. and Hoenger, A. (2004). Modulation of kinesin binding by the C-termini of tubulin. EMBO J. 23, 989-999.

Thompson, H. M., Cao, H., Chen, J., Euteneuer, U. and McNiven, M. A. (2004). Dynamin 2 binds g-tubulin and participates in centrosome cohesion. Nat. Cell Biol. 6, 335-342.

Zhang, Y., Li, N., Caron, C., Matthias, G., Hess, D., Khochbin, S. and Matthias, P. (2003). HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J. 22, 1168-1179.