Tubulin protein, frozen (>99% pure) (T238)

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)).

This product is also available 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.

T238 is identical to T237 when 10% glycerol is added. >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.

Biological Activity
The biological activity of T238 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. 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. See Fig 2 on TL238 page for example polymerization results.

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. See Figure 2 on product page for tubulin plus glycerol (Cat. # T237) for polymerization in the presence of glycerol and paclitaxel.

References

Shelanski, M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768

Examples of publications where this product was used
Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A. et al. (1998). Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282, 1914-1917.

King, S. J. and Schroer, T. A. (2000). Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20-24.

Melander Gradin, H., Larsson, N., Marklund, U. and Gullberg, M. (1998). Regulation of microtubule dynamics by extracellular signals: cAMP-dependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J. Cell Biol. 140, 131-141.

Sosa, H., Dias, D. P., Hoenger, A., Whittaker, M., Wilson-Kubalek, E., Sablin, E., Fletterick, R. J., Vale, R. D. and Milligan, R. A. (1997). A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis. Cell 90, 217-224.