Product Uses Include
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 porcine 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.
This kit contains enough materials for 24 assays (BK006P). The following reagents are included:
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.
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 firstname.lastname@example.org
|Ana, Gloria et al.||Synthesis and biological evaluation of 1‐(Diarylmethyl)‐1h‐1,2,4‐triazoles and 1‐(diarylmethyl)‐1h‐imidazoles as a novel class of anti‐mitotic agent for activity in breast cancer||Pharmaceuticals||2021||ISSN 1424-8247|
|Ibrahim, Tarek S. et al.||Discovery of novel quinoline-based analogues of combretastatin A-4 as tubulin polymerisation inhibitors with apoptosis inducing activity and potent anticancer effect||Journal of Enzyme Inhibition and Medicinal Chemistry||2021||ISSN 1475-6374|
|Malebari, Azizah M. et al.||Synthesis and antiproliferative evaluation of 3‐chloroazetidin‐2‐ones with antimitotic activity: Heterocyclic bridged analogues of combretastatin a‐4||Pharmaceuticals||2021||ISSN 1424-8247|
|Khayyat, Ahdab N. et al.||Design, synthesis, and antipoliferative activities of novel substituted imidazole-thione linked benzotriazole derivatives||Molecules||2021||ISSN 1420-3049|
|Malebari, Azizah M. et al.||β-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells||European Journal of Medicinal Chemistry||2020||ISSN 1768-3254|
|Ibrahim, T S et al.||Potent quinoline-containing combretastatin a-4 analogues: design, synthesis, antiproliferative, and anti-tubulin activity||Pharmaceuticals||2020||Article Link|
|Schneidewind, Tabea et al.||The Pseudo Natural Product Myokinasib Is a Myosin Light Chain Kinase 1 Inhibitor with Unprecedented Chemotype||Cell Chemical Biology||2019||ISSN 2451-9448|
|Wang, Shu et al.||3-Vinylazetidin-2-Ones: Synthesis, antiproliferative and tubulin destabilizing activity in MCF-7 and MDA-MB-231 Breast Cancer Cells||Pharmaceuticals||2019||ISSN 1424-8247|
|Malebari, Azizah M. et al.||β-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells||European Journal of Medicinal Chemistry||2017||ISSN 1768-3254|
|Majellaro, Maria et al.||Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl Nicotinamides||ChemMedChem||2017||ISSN 1860-7187|
|Chan, Eddie et al.||The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells||Biochimica et Biophysica Acta - Molecular Cell Research||2016||ISSN 1879-2596|
|Ayoub, Ahmed Taha et al.||Antitumor Activity of Lankacidin Group Antibiotics Is Due to Microtubule Stabilization via a Paclitaxel-like Mechanism||Journal of Medicinal Chemistry||2016||ISSN 1520-4804|
|Tsai, An Chi et al.||In vitro and in vivo anti-tumour effects of MPT0B014, a novel derivative aroylquinoline, and in combination with erlotinib in human non-small-cell lung cancer cells||British Journal of Pharmacology||2014||ISSN 0007-1188|
|Tsai, A. C. et al.||Orally active microtubule-targeting agent, MPT0B271, for the treatment of human non-small cell lung cancer, alone and in combination with erlotinib||Cell Death & Disease||2014||ISSN 2041--4889|
|Jackson, Steven J.T. et al.||Curcumin binds tubulin, induces mitotic catastrophe, and impedes normal endothelial cell proliferation||Food and Chemical Toxicology||2013||ISSN 0278-6915|
|Brachmann, Saskia M. et al.||Characterization of the mechanism of action of the pan class i PI3K inhibitor NVP-BKM120 across a broad range of concentrations||Molecular Cancer Therapeutics||2012||ISSN 1535-7163|
|Chen, Chun Han et al.||Protopine, a novel microtubule-stabilizing agent, causes mitotic arrest and apoptotic cell death in human hormone-refractory prostate cancer cell lines||2012||PMID 22033245|
|Hartley, R. M. et al.||Polygamain, a New Microtubule Depolymerizing Agent That Occupies a Unique Pharmacophore in the Colchicine Site||Molecular Pharmacology||2012||ISSN 0026-895X|
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|Geng, Feng et al.||Allyl isothiocyanate arrests cancer cells in mitosis, and mitotic arrest in turn leads to apoptosis via Bcl-2 protein phosphorylation||Journal of Biological Chemistry||2011||ISSN 0021-9258|
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|Chang, Chia Hao et al.||Mycotoxin citrinin induced cell cycle G2/M arrest and numerical chromosomal aberration associated with disruption of microtubule formation in human cells||Toxicological sciences : an official journal of the Society of Toxicology||2011||ISSN 1096--0929|
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Question 1: What is the difference between this kit and BK004P?
Answer 1: Both the BK004P and BK006P are tubulin polymerization kits that are absorbance-based rather than fluorescence-based. The only difference between the two absorbance-based kits is that BK004P uses 97% pure tubulin (remaining 3% are MAPs) while BK006P uses >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 such as glycerol or taxol with as little as 3 or 4 mg/ml tubulin using the BK004P kit. In this case MAPs act as polymerization enhancers. With BK006P, 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. In some cases, glycerol can interfere with the binding of tubulin accessory proteins or compounds. Assay conditions can easily be altered to test for glycerol interference.
Question 2: Which kit is best for screening a compound/reagent/drµg for its 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/drµg screening, we recommend the absorbance-based tubulin polymerization assay BK004P. 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/reagents/drµgs. 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, thus BK011P is the most economical when requiring >30 assays for the project.
If you have any questions concerning this product, please contact our Technical Service department at email@example.com