Tubulin polymerization assay using >99% pure tubulin, OD based - Porcine (BK006P)

Product Uses Include

1. Screening compounds for effects on tubulin polymerization activity.
2. Screening proteins for effects on tubulin polymerization activity.
3. Determining that tubulin alone (not MAPs) is the target for compounds that affect microtubule polymerization.
4. Teaching aid for undergraduate/graduate class in pharmacology.
   
   

Introduction
This assay is based on an adaptation of the original method of Shelanski et al. and Lee et al. (1,2), which demonstrated that light is scattered by microtubules to an extent that is proportional to the concentration of microtubule polymer. The resulting polymerization curve is representative of the three phases of microtubule polymerization, namely nucleation, growth and steady state equilibrium. See the About Tubulin page for more information. The assay is optimized for a 96-well format for low CVs and efficient sample handling.

This kit contains the bovine neuronal tubulin of the highest available purity (>99% pure, Cat. # T240). The same type of assay is also available with HTS tubulin (Cat. # BK004P) and can serve as an economical, but slightly less sensitive alternative to BK006P. HTS tubulin (Cat. # HTS03) is >97% pure. Cytoskeleton, Inc. also provides a fluorescence based tubulin polymerization assay in miniaturized format (Cat. # BK011P) making it ideal for high throughput screening.

If you are interested in using either of these tubulin polymerization assays in a high throughput setting, please contact our technical service department for advice and bulk quotes.

Kit contents
This kit contains enough materials for 24 assays (BK006P). The following reagents are included:

1. Tubulin, >99% pure, lyophilized (Cat. # T240)
2. GTP solution (Cat. # BST06)
3. General tubulin buffer (PEM, Cat. # BST01)
4. Tubulin glycerol buffer (Cat. # BST05)
5. Paclitaxel positive control (Cat. # TXD01)
6. DMSO for paclitaxel.
7. Half area 96-well plate for polymerization reactions
8. Manual with detailed protocols and extensive troubleshooting guide.
   
   

Equipment needed

1. 96-well plate spectrophotometer with filters to read optical density at 340 nm.
2. Multi-channel pipette for rapid pipetting of tubulin
   
   

Example results
The BK006P kit was used to study the effects of Paclitaxel, a polymerization enhancer and Nocodazole, a polymerization inhibitor on tubulin polymerization (Fig. 1)

	Figure 1. Tubulin polymerization curves from kit BK006P. The figure shows a standard polymerization curve (Control curve) containing a 100 µl volume of 3 mg/ml tubulin in 80 mM PIPES pH 7.0, 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP and 10% glycerol. Polymerization was started by incubation at 37°C and followed by absorption readings at 340 nm. Under these conditions, polymerization Vmax is enhanced 4 fold in the presence of 10 µM paclitaxel and reduced 5.5 fold in the presence of 10 µM nocodazole.

References

1. Shelanski, M. L., Gaskin, F. and Cantor, C. R. (1973). Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. U.S.A. 70, 765-768.
2. Lee, J. C. and Timasheff, S. N. (1977). In vitro reconstitution of calf brain microtubules: effects of solution variable. Biochemistry, 16, 1754-1762.

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

•   For our Tubulin Polymerization (Absorbance) Excel Template please download here.
•   For our IC50 from Vmax Polymerization Excel Template please download here.

Product Description

MSDS

Datasheet

Cat #

Tubulin polymerization HTS assay using >99% pure tubulin, OD based - Porcine (BK006P) BK006P

O'Boyle NM, Carr M, Greene LM, Bergin O, Nathwani SM, McCabe T, Lloyd DG, Zisterer DM, Meegan MJ. (2010). Synthesis and Evaluation of Azetidinone Analogues of Combretastatin A-4 as Tubulin Targeting Agents.. J Med Chem.

Jacob Kushkuley, Walter K. H. Chan, Sangmook Lee, Joel Eyer, Jean-Francois Leterrier, Franck Letournel and Thomas B. Shea (2009). Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules. J Cell Science 122 ,3579-86.

Chen, Z., Merta, P. J., Lin, N. H., Tahir, S. K., Kovar, P., Sham, H. L. and Zhang, H. (2005). A-432411, a novel indolinone compound that disrupts spindle pole formation and inhibits human cancer cell growth. Mol. Cancer Ther. 4, 562-568.

Huang, Y. T., Huang, D. M., Guh, J. H., Chen, I. L., Tzeng, C. C. and Teng, C. M. (2005). CIL-102 interacts with microtubule polymerization and causes mitotic arrest following apoptosis in the human prostate cancer PC-3 cell line. J. Biol. Chem. 280, 2771-2779.

Rouzier, R., Rajan, R., Wagner, P., Hess, K. R., Gold, D. L., Stec, J., Ayers, M., Ross, J. S., Zhang, P., Buchholz, T. A. et al. (2005). Microtubule-associated protein tau: A marker of paclitaxel sensitivity in breast cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 8315-8320.

Jiang, J. D., Denner, L., Ling, Y. H., Li, J. N., Davis, A., Wang, Y., Li, Y., Roboz, J., Wang, L. G., Perez-Soler, R. et al. (2002). Double blockade of cell cycle at G1-S transition and M phase by 3-iodoacetamido benzoyl ethyl ester, a new type of tubulin ligand. Cancer Res. 62, 6080-6088.

Mooberry, S. L., Tien, G., Hernandez, A. H., Plubrukarn, A. and Davidson, B. S. (1999). Laulimalide and isolaulimalide, new paclitaxel-like microtubule-stabilizing agents. Cancer Res. 59, 653-660.

Coming soon!   If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com