Product Uses Include
- Cost effective high throughput screen for anti-cancer compounds.
- Basic research to measure compound IC50s and specificity for tubulin.
- Screening proteins for effects on tubulin polymerization activity.
- Teaching aid for undergraduate/graduate class in pharmacology.
IntroductionThis assay is an economical one step procedure for determining the effects of drugs or proteins on tubulin polymerization. It is an adaptation of an assay originally described by Bonne, D. et al. (1). Polymerization is followed by fluorescence enhancement due to the incorporation of a fluorescent reporter into microtubules as polymerization occurs. The standard assay uses neuronal tubulin (Cat. # T240), which generates a polymerization curve representing the three phases of microtubule formation; namely nucleation, growth and steady state equilibrium. Other tubulins, such as HeLa cell tubulin (Cat. # H001) can also be used in this assay. The low volume per assay of 50 µl and the low tubulin concentrations, 2 mg/ml final concentration (100 µg per assay), makes this an ideal choice for studying the more expensive cancer cell tubulin reagents and for high throughput applications.
The classic tubulin polymerization assay uses absorbance readings at 340 nm to follow microtubule formation. It is based upon the fact that light is scattered by microtubules to an extent that is proportional to the concentration of microtubule polymer. This assay is offered by Cytoskeleton, Inc. (Cat. # BK006P). The fluorescence based assay has been compared to the absorbance based format and the comparisons are given in Table 1 below. For help in selecting the best assay format for your needs, contact tservice@cytoskeleton.com.
Table 1. Comparison of Fluorescence versus Absorbance Based Polymerization Assays
| Assay Characteristics | Absorbance Assay | Fluorescence Assay |
| Tubulin used per assay | 300 µg | 100 µg |
| Volume of reaction | 100 µl | 50 µl |
| Signal to noise ratio (S/N) | 2 | 4 |
| Coefficient of variation (cv)* | 13% | 11% |
| Paclitaxel EC50** | 1 µM | 1 µM |
| Vinblastine IC50 ** | 0.6 µM | 0.6 µM |
| Possible problems | Glycerol in standard assay format may interfere with drug or protein binding. Assay conditions can easily be altered to test this. | Fluorescent reporter may interfere with drug or protein binding. |
| *: Duplicate samples**: Under standard assay conditions. Conditions can be optimized for polymerization enhancers or inhibitors. | ||
Kit contentsThe kit contains sufficient material for 96 assays in 50 µl format. The following components are included:
- >99% pure tubulin (Cat. # T240)
- General tubulin buffer with fluorescent reporter
- Microtubule glycerol buffer (Cat. # BST05)
- GTP solution (Cat. # BST06)
- Paclitaxel positive control (Cat. # TXD01)
- Half area 96-well plate. Black, flat bottom
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- Tempererature controlled 96-well plate fluorimeter equipped with filters for exitation at 340-360 nm and emission at 420-450 nm
Example resultsCompounds or proteins that interact with tubulin will often alter one or more of the characteristic phases of polymerization. For example, Figure 1 shows the effect of adding the anti-mitotic drug paclitaxel to the standard polymerization reaction. A 3 µM concentration of paclitaxel eliminates the nucleation phase and enhances the Vmax of the growth phase. Thus, one application of this assay is the identification of novel anti-mitotics. Figure 1 also shows the effect of adding the microtubule destabilizing drug, vinblastine. At 3 µM final concentration, vinblastine causes a drastic decrease in Vmax and reduction in final polymer mass.
Figure 1. Tubulin polymerization using the fluorescence based tubulin polymerization assay (BK011P). Tubulin was incubated alone (Control), with Paclitaxel or Vinblastine. Each condition was tested in duplicate. Polymerization was measured by excitation at 360 nm and emission at 420 nm. The three Phases of tubulin polymerization are marked for the control polymerization curve; I: nucleation, II: growth, III: steady state equillibrium.
References
Bonne, D., Heusele, C., Simon, C., and Pantaloni, D. (1985). 4’, 6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and mictrotubules. J. Biol. Chem. 260, 2819-2825.
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 (Fluorescence) Excel Template please download here.- For our IC50 from Vmax Polymerization Excel Template please download here.
Baker, S. J. et al. (2020). A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent. Molecular cell, 79(1), 180-190.e4. https://doi.org/10.1016/j.molcel.2020.05.024
La, T. M. et al. Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes. FASEB J. n/a, fj.202001240RR (2020).
Lemjabbar-Alaoui, H., Peto, C. J., Yang, Y.-W. & Jablons, D. M. AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers. Am. J. Cancer Res. 10, 2649–2676 (2020).
Chen, S.-Y. et al. Exosomal 2′,3′-CNP from mesenchymal stem cells promotes hippocampus CA1 neurogenesis/neuritogenesis and contributes to rescue of cognition/learning deficiencies of damaged brain. Stem Cells Transl. Med. 9, 499–517 (2020).
Zhernov, I., Diez, S., Braun, M. & Lansky, Z. Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional Diversity. Curr. Biol. 30, 3342-3351.e5 (2020).
Lemjabbar-Alaoui, H., Peto, C. J., Yang, Y.-W. & Jablons, D. M. AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers. Am. J. Cancer Res. 10, 2649–2676 (2020).
La, T. M. et al. Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes. FASEB J. n/a, fj.202001240RR (2020).
Liu, Qian et al. “Identification of a lathyrane-type diterpenoid EM-E-11-4 as a novel paclitaxel resistance reversing agent with multiple mechanisms of action.” Aging vol. 12,4 (2020): 3713-3729. doi:10.18632/aging.102842
.-H. Choi et al. 2014. Suprafenacine, an indazole-hydrazide agent, targets cancer cells through microtubule destabilization. PLoS ONE. 9, e110955.
S. Senese et al. 2014. Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development. Cell Death and Disease. 5, e1462.
M. Mei et al. 2014. A new 2a,5a,10b,14b-tetraacetoxy-4(20),11-taxadiene (SIA) derivative overcomes paclitaxel resistance by inhibiting MAPK signaling and increasing paclitaxel accumulation in breast cancer cells. PLoS ONE. 9, e104317.
I.M. Fawzy et al. 2014. Newly designed and synthesized curcumin analogs with in vitro cytotoxicity and tubulin polymerization activity. Chem. Biol. Drug Des. DOI: 10.1111/cbdd.12464.
W.M. Remers et al. 2014. Synthesis and antitumor activity of heterocycles related to carbendazim. J. Heterocycl. Chem. DOI: 10.1002/jhet.1976.
T. Shigehiro et al. 2014. Efficient drug delivery of paclitaxel glycoside: A novel solubility gradient encapsulation into liposomes coupled with immunoliposomes preparation. PLoS ONE. 9, e107976.
Sidhaye et al., 2012. A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and Stability. PLoS ONE 7: e38717.
Zach et al., 2012. The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule association. Hum. Mol. Genet. doi: 10.1093/hmg/dds268.
Dyrager et al., 2011. Inhibitors and promoters of tubulin polymerization: Synthesis and biological evaluation of chalcones and related dienones as potential anticancer agents. Bioorg. Med. Chem. v 19, pp 2659-2665.
Hwang et al., 2011. Induction of tubulin polymerization and apoptosis in malignant mesothelioma cells by a new compound JBIR-23. Cancer Lett. v 300, pp 189-196.
Kim et al., 2011. Zinc stimulates tau S214 phosphorylation by the activation of Raf/mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase pathway. Neuroreport, v22, pp 839-844.
For the most recent publications citing this product, please contact our Technical Service department at tservice@cytoskeleton.com
Question 1: What are the advantages to using this kit?
Answer 1: BK011P is a fluorescence-based tubulin polymerization assay kit. Compared to the absorbance-based kits, BK011P has increased sensitivity, signal-to-noise ratio and an improved coefficient of variation. The greater sensitivity allows the researcher to use 1/3 as much tubulin which means that the BK011P kit provides 96 assays versus the 30 assays of BK004P or BK006P. On a cost per assay basis, BK011P is the best value of the tubulin polymerization kits.
Question 2: Which kit is best for screening a compound for it’s effects on tubulin polymerization?
Answer 2: All 3 tubulin polymerization kits (2 absorbance-based kits, BK004P and BK006P; 1 fluorescence-based kit, BK011P) are well-suited for screening of potential tubulin polymerization enhancers and inhibitors. Each kit has its own pros and cons. For initial compound/drug screening, we recommend the absorbance-based tubulin polymerization assay BK004P which is the most economical. This kit uses 97% pure tubulin (remaining 3% are MAPs) while BK006P and BK011P use >99% pure tubulin. This is an important difference because the presence of MAPs means that tubulin polymerization can be examined in the absence of enhancers or inhibitors with as little as 3 or 4 mg/ml tubulin using the BK004P kit. To study enhancers, we recommend using 3 mg/ml tubulin, whereas 4 mg/ml tubulin is recommended for inhibitors. In the case of BK004P, MAPs act as polymerization enhancers. With BK006P and BK011P, an enhancer such as glycerol or taxol must be used to drive tubulin polymerization with concentrations <5 mg/ml tubulin. Using tubulin at 5 mg/ml or higher allows for the omission of glycerol or taxol, but requires additional tubulin. In some cases, glycerol can interfere with the binding of tubulin accessory proteins or compounds. However, since BK011P is fluorescence-based, there is increased sensitivity that allows the researcher to use 1/3 as much tubulin with greater sensitivity. Thus, the kit provides 96 assays versus the 30 assays of BK004P or BK006P. Assay conditions can easily be altered to test for glycerol interference.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
ebiomall.com
