Cat. # T237

Product Uses Include

• IC50 & EC50 determinations for anti-tubulin ligands.
• Microtubule 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 at 10 mg/ml as a frozen liquid in G-PEM (General tubulin buffer (Cat. # BST01) with 1 mM GTP (Cat. # BST06)) plus 5% glycerol (Cat. # BST05).

This product is also available without glycerol in a lyophilized, fully active format (Cat. # TL238). The lyophilized format tubulin has many advantages over the frozen format. TL238 costs less and since it allows for ambient temperature shipping, shipping is also significantly less costly. In addition, the lyophilized format allows for simpler (+4°C desiccated vs -70°C) and longer storage. When TL238 is reconstituted to 10 mg.ml in G-PEM plus 5% glycerol, it is identical to T237.

>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.

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

Figure 1: 100 µg of T237 was run on a 10% SDS-PAGE gel and stained with Coomassie Blue.

Biological Activity
One unit of tubulin is defined as 5.0 mg of purified protein (as determined by the Precision Red Advanced Protein Assay Reagent cat. # ADV02). When tubulin at a concentration of 1.0 unit/ml in G-PEM buffer plus 10% glycerol [v/v] is incubated at 35°C for 30 min, an increase in optical density at 340 nm from 0.0 to 1.0 is observed. This corresponds to that more than 95% of the tubulin has polymerized. The average number of MTs per ml under these conditions is 1.0x10¹² and the mean length of the microtubule population is approximately 10 µm as measured by electron microscopy.

Figure 2. Tubulin polymerization in 96-well format using tubulin plus glycerol. T237 alone (sample 1), T237 plus 3 µM paclitaxel (cat. # TXD01) (sample 2) or T237 plus 3 µM nocodazole (sample 3) was incubated at 37°C in a 1/2 area 96-wellplate and polymerization was measured as a function of the optical density at 340 nm.

Examples of publications where this product was used
D'Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V. M., Bird, T. D. and Schellenberg, G. D. (1999). Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. Proc. Natl. Acad. Sci. U. S. A. 96, 5598-5603.

Korinek, W. S., Copeland, M. J., Chaudhuri, A. and Chant, J. (2000). Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 287, 2257-2259.

Murphy, S. M., Urbani, L. and Stearns, T. (1998). The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p. J. Cell Biol. 141, 663-674.

Shan, B., Medina, J. C., Santha, E., Frankmoelle, W. P., Chou, T. C., Learned, R. M., Narbut, M. R., Stott, D., Wu, P., Jaen, J. C. et al. (1999). Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors. Proc. Natl. Acad. Sci. U. S. A. 96, 5686-5691.

Tai, A. W., Chuang, J. Z., Bode, C., Wolfrum, U. and Sung, C. H. (1999). Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. Cell 97, 877-887.