Tubulin ligand competition assay (CDS15)

Product Uses Include

• Screening compounds for effects on tubulin polymerization activity.
• Screening proteins for effects on tubulin polymerization activity.
• Teaching aid for undergraduate/graduate class in pharmacology.
• Economical approach to HTS with tubulin.
  
  

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 large sample number handling.

This kit contains "HTS tubulin", which is >97% pure (Cat. # HTS02). The same assay is also available with our highest purity tubulin (>99% pure, Cat. # TL238) and is sold under Cat. # BK006. BK004 provides an economical alternative to the higher purity BK006 assay and can be a good choice for large primary screens. Cytoskeleton, Inc. also provides a fluorescence based tubulin polymerization assay in miniaturized format (Cat. # BK011P), which is also 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 (BK004). The following reagents are included:

1. HTS tubulin, lyophilized (Cat. # HTS02)
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 BK004 kit was used to study the effect of 48 different conditions on tubulin polymerization rates in a 96 well format. Each condition was tested in duplicate samples (Fig. 1)

Figure 1. Tubulin polymerization in 96-well format using BK004. 48 different tubulin polymerization conditions were tested in duplicates. Wells A1 and A2 represent a control reaction and wells C1 and C2 represent the addition of the microtubule stabilizing drug Paclitaxel, resulting in an increased polymerization rate. Each well represents one polymerization reaction. The x-axis is 20 min and the y-axis corresponds to 0.0-0.2 OD at 340 nm.

 

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

Product Description

MSDS

Datasheet

Cat #

Tubulin ligand competition assay (CDS15) CDS15

Davis, P. D., Dougherty, G. J., Blakey, D. C., Galbraith, S. M., Tozer, G. M., Holder, A. L., Naylor, M. A., Nolan, J., Stratford, M. R., Chaplin, D. J. et al. (2002). ZD6126: a novel vascular-targeting agent that causes selective destruction of tumor vasculature. Cancer Res. 62, 7247-7253.

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