Material
Tubulin and microtubule associated proteins (MAPs) has been purified from bovine brain by an adaptation of the method of Shelanski et al. (1). Tubulin is supplied as a white lyophilized powder. The protein composition is approximately 70% tubulin (55 kDa heterodimer) and 30% MAPs (see Figure 1). The MAPs in this product act to stabilize microtubules and to enhance tubulin polymerization. MAP rich tubulin can polymerize efficiently at 1-2 mg/ml.
MAP-rich tubulin is also available from a porcine source (Cat. # ML116)
Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are approximately 70% tubulin and 30% MAPs.
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Figure 1: 100 µg of ML113 was run on a 10% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02). |
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Biological Activity
One unit of ML113 is defined as 5.0 mg of protein (as determined by the Precision Red Protein Assay Reagent (cat. # ADV02). MAP-rich tubulin will polymerize efficiently at protein concentrations above 1 mg/ml and temperatures above 24°C.
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Figure 2: MAP-rich tubulin polymerization under different conditions. Samples of ML113 in G-PEM buffer were placed in 96-well plates and incubated at the specified temperatures. Sample 1: 2 mg/ml at 37°C. Sample 2: 2 mg/ml at 24°C. Sample 3: 1 mg/ml at 24°C. Sample 4, same as 3 except in the presence of 3 µM nocodazole. Tubulin polymerization was observed by measuring the optical density at 340 nm over time. |
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References
Shelanski M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Product Description
MSDS
Datasheet
Cat #
Tubulin protein: MAP rich: bovine brain
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ML113
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Kuo, C. C., Hsieh, H. P., Pan, W. Y., Chen, C. P., Liou, J. P., Lee, S. J., Chang, Y. L., Chen, L. T., Chen, C. T. and Chang, J. Y. (2004). BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res. 64, 4621-4628.
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.
Xiao, D., Pinto, J. T., Soh, J. W., Deguchi, A., Gundersen, G. G., Palazzo, A. F., Yoon, J. T., Shirin, H. and Weinstein, I. B. (2003). Induction of apoptosis by the garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH2-terminal kinase 1 activation. Cancer Res. 63, 6825-6837.
Xie, Z., Sanada, K., Samuels, B. A., Shih, H. and Tsai, L. H. (2003). Serine 732 phosphorylation of FAK by Cdk5 is important for microtubule organization, nuclear movement, and neuronal migration. Cell 114, 469-482.
Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J. and Olsen, R. W. (1999). GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton. Nature 397, 69-72.
Question 1: What is the difference between 70% pure bovine brain tubulin (Cat. # ML113) and 70% pure porcine brain tubulin (Cat. # ML116)?
Answer 1: The only difference is the source of the tubulin: bovine vs porcine brains. Functionally, there are no significant differences between tubulin isolated from bovine brains vs porcine brains. Stringent quality control and in-house testing has revealed no differences in polymerization, response to drugs (enhancers and inhibitors) or binding to accessory proteins (e.g., motor proteins). (See Comparison of Porcine and Bovine tubulin)
Question 2: Does tubulin polymerization using 70% pure bovine brain tubulin/30% MAPs (Cat. # ML113) require additional enhancers such as glycerol or taxol?
Answer 2: No additional enhancers are required when using 2 mg/ml of the MAP-enriched 70% pure tubulin. The recommended polymerization reaction using 70% pure tubulin contains 180 μl of 2 mg/ml tubulin in 80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCl2 and 1 mM GTP. Polymerization is started by incubation at 37°C and followed by absorption readings at 340 nm. Under these conditions polymerization reaches a maximal OD340 between 0.3 - 0.4 within 20 minutes. In this experimental set up (180 μl volume in a spectrophotometer with a pathlength of 0.8 cm using Corning Costar’s half area well plate Cat. # 3696) an OD340 of 0.16 is approximately equal to 1 mg per ml of tubulin polymer mass. Thus, under the conditions described, >90% of the tubulin is polymerized. If polymerization enhancers such as taxol are to be used or tested, we recommend reducing the tubulin concentration to 1 mg/ml. These conditions will result in a more pronounced nucleation phase and a lower polymerization curve.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
