Tubulin polymerization assay using >99% pure tubulin, fluorescence based (BK011P)

Tubulin polymerization HTS assay using >99% pure tubulin, fluorescence based (BK011P)
$0.00

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.

Introduction
This 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 CharacteristicsAbsorbance AssayFluorescence 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 problemsGlycerol 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 contents
The kit contains sufficient material for 96 assays in 50 µl format. The following components are included:

  1. >99% pure tubulin (Cat. # T240)
  2. General tubulin buffer with fluorescent reporter
  3. Microtubule glycerol buffer (Cat. # BST05)
  4. GTP solution (Cat. # BST06)
  5. Paclitaxel positive control (Cat. # TXD01)
  6. Half area 96-well plate. Black, flat bottom
  7. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. Tempererature controlled 96-well plate fluorimeter equipped with filters for exitation at 340-360 nm and emission at 420-450 nm

Example results
Compounds 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. 

bk011fig1

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.
AuthorTitleJournalYearArticle Link
Kumar N, Manoj et al.Benzothiazole-[1,2,3]triazolo[5,1-a]isoindoles: Synthesis, anticancer activity, bioavailability and in silico studies against Gama-Tubulin proteinJournal of Molecular Structure2022ISSN 0022-2860
Yao, Yongfang et al.Angiogenesis and anti-leukaemia activity of novel indole derivatives as potent colchicine binding site inhibitorsJournal of Enzyme Inhibition and Medicinal Chemistry2022ISSN 1475-6374
Wang, Zhan et al.Design, synthesis and biological evaluation of colchicine glycoconjugates as tubulin polymerization inhibitorsBioorganic and Medicinal Chemistry2022ISSN 1464-3391
Ma, Juan et al.Discovery of Novel 3,4-Dihydro-2(1H)-Quinolinone Sulfonamide Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer ActivityMolecules2022ISSN 1420-3049
Würtz, Martin et al.Modular assembly of the principal microtubule nucleator γ-TuRCNature Communications2022ISSN 2041-1723
Liu, Chao et al.Synthesis and biological evaluation of BU-4664L derivatives as potential anticancer agentsBioorganic and Medicinal Chemistry Letters2022ISSN 1464-3405
Wang, Guangcheng et al.Design, synthesis and biological evaluation of novel 2-phenyl-4,5,6,7-tetrahydro-1H-​indole derivatives as potential anticancer agents and tubulin polymerization inhibitorsArabian Journal of Chemistry2022ISSN 1878-5352
Patel, Onisha et al.Structural basis for small molecule targeting of Doublecortin Like Kinase 1 with DCLK1-IN-1Communications Biology2021ISSN 2399-3642
Fareed, Momen R. et al.New multi-targeted antiproliferative agents: Design and synthesis of ic261-based oxindoles as potential tubulin, ck1 and egfr inhibitorsPharmaceuticals2021ISSN 1424-8247
Rahimzadeh Oskuei, Sara et al.Design, synthesis and biological evaluation of novel imidazole-chalcone derivatives as potential anticancer agents and tubulin polymerization inhibitorsBioorganic Chemistry2021ISSN 1090-2120
Liang, Dong et al.Identification of anthelmintic parbendazole as a therapeutic molecule for HNSCC through connectivity map-based drug repositioningActa Pharmaceutica Sinica B2021ISSN 2211-3843
Sun, Chiao Yin et al.LMBD1 protein participates in cell mitosis by regulating microtubule assemblyBiochemical Journal2021ISSN 1470-8728
Wang, Chao et al.Design, synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule destabilizersJournal of Enzyme Inhibition and Medicinal Chemistry2021ISSN 1475-6374
Würtz, Martin et al.Reconstitution of the recombinant human γ-tubulin ring complexOpen Biology2021ISSN 2046-2441
Grohmann, Christoph et al.Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumorsCell Death and Disease2021ISSN 2041-4889
Yang, Mei et al.C118P, a novel microtubule inhibitor with anti-angiogenic and vascular disrupting activities, exerts anti-tumor effects against hepatocellular carcinomaBiochemical Pharmacology2021
Lead optimization of novel quinolone chalcone compounds by a structure-activity relationship (SAR) study to increase efficacy and metabolic stability2021Article Link
Shawky, Ahmed M. et al.Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety: design, synthesis, molecular docking, and biological evaluation as potential multi-target cytotoxic agentsJournal of Enzyme Inhibition and Medicinal Chemistry2021ISSN 1475-6374
Woo, Jung AA et al.β-arrestin1 promotes tauopathy by transducing GPCR signaling, disrupting microtubules and autophagyLife science alliance2021ISSN 2575--1077
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 actionAging2020ISSN 1945-4589
Abdel-Rahman, Somaya A. et al.Cyclohepta[ b]thiophenes as Potential Antiproliferative Agents: Design, Synthesis, in Vitro, and in Vivo Anticancer EvaluationACS Pharmacology and Translational Science2020ISSN 2575-9108
Barnes, Natalie G. et al.A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitorsBioorganic and Medicinal Chemistry2020ISSN 1464-3391
Palumbo, Valeria et al.Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerizationJournal of Cell Science2020ISSN 1477-9137
Mirzaei, Salimeh et al.Synthesis, structure-activity relationship and molecular docking studies of novel quinoline-chalcone hybrids as potential anticancer agents and tubulin inhibitorsJournal of Molecular Structure2020ISSN 0022-2860
Chen, Shih Yin 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 brainStem Cells Translational Medicine2020ISSN 2157-6580
Song, Ming Yu et al.Exploring diverse-ring analogues on combretastatin a4 (Ca-4) olefin as microtubule-targeting agentsInternational Journal of Molecular Sciences2020ISSN 1422-0067
Ullah, Imran et al.An Antiparasitic Compound from the Medicines for Malaria Venture Pathogen Box Promotes Leishmania Tubulin PolymerizationACS Infectious Diseases2020ISSN 2373-8227
Tariq, Ammarah et al.In vitro reconstitution of branching microtubule nucleationeLife2020ISSN 2050-084X
Zhernov, Ilia et al.Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional DiversityCurrent Biology2020ISSN 1879-0445
Zdioruk, Mykola et al.A new inhibitor of tubulin polymerization kills multiple cancer cell types and reveals p21-mediated mechanism determining cell death after mitotic catastropheCancers2020ISSN 2072-6694
Skidmore, Lillian et al.ARX788, a site-specific anti-HER2 antibody–drug conjugate, demonstrates potent and selective activity in HER2-low and T-DM1–resistant breast and gastric cancersMolecular Cancer Therapeutics2020ISSN 1538-8514
Wang, Yanming et al.Antibody-drug conjugate using ionized CYS-linker-mmae as the potent payload shows optimal therapeutic safetyCancers2020ISSN 2072-6694
Baker, Stacey J. et al.A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding AgentMolecular Cell2020ISSN 1097-4164
Morita, Ken et al.Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2Cell2020ISSN 1097-4172
Dillon, Gregory M. et al.Acute inhibition of the CNS-specific kinase TTBK1 significantly lowers tau phosphorylation at several disease relevant sitesPLoS ONE2020ISSN 1932-6203
Zhai, Min'an et al.3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors: Rational design, synthesis and biological evaluationEuropean Journal of Medicinal Chemistry2019ISSN 1768-3254
Su, Wenhui et al.Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganizationFASEB journal : official publication of the Federation of American Societies for Experimental Biology2019ISSN 1530-6860
Wang, Yanming et al.Novel silyl ether-based acid-cleavable antibody-MMAE conjugates with appropriate stability and efficacyCancers2019ISSN 2072-6694
Cui, Ying Jie et al.Synthesis of novel pyrazole derivatives and their tumor cell growth inhibitory activityMolecules2019ISSN 1420-3049
Shaik, Thoukhir B. et al.Evaluation of Anticancer and Anti-Mitotic Properties of Quinazoline and Quinazolino-Benzothiadiazine DerivativesAnti-Cancer Agents in Medicinal Chemistry2019ISSN 1871-5206
Sonawane, Vinay et al.Cink4T, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapyEuropean Journal of Medicinal Chemistry2019ISSN 1768-3254
Xia, Xiaoyu et al.Leukemia Cell Cycle Chemical Profiling Identifies the G2-Phase Leukemia Specific Inhibitor Leusin-1ACS Chemical Biology2019ISSN 1554-8937
Behbahani, Fatemeh Shaebani et al.Synthesis and biological evaluation of novel benzo[c]acridine-diones as potential anticancer agents and tubulin polymerization inhibitorsArchiv der Pharmazie2019ISSN 1521-4184
Karimikia, Ehsan et al.Colchicine-like β-acetamidoketones as inhibitors of microtubule polymerization: Design, synthesis and biological evaluation of in vitro anticancer activityIranian Journal of Basic Medical Sciences2019ISSN 2008-3874
Patterson, Jesse C. et al.VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer CellsCell Systems2019ISSN 2405-4720
Mao, Bai Ping et al.CaMSAP2 is a microtubule minus-end targeting protein that regulates BTB dynamics through cytoskeletal organizationEndocrinology2019ISSN 1945-7170
Eberle-Singh, Jaime A. et al.Effective delivery of a microtubule polymerization inhibitor synergizes with standard regimens in models of pancreatic ductal adenocarcinomaClinical Cancer Research2019ISSN 1557-3265
Lukinavičius, Gražvydas et al.Fluorescent dyes and probes for super-resolution microscopy of microtubules and tracheoles in living cells and tissuesChemical Science2018ISSN 2041-6539
Tian, Zhenhua et al.Biological activity and interaction mechanism of the diketopiperazine derivatives as tubulin polymerization inhibitorsRSC Advances2018ISSN 2046-2069
Simpkins, Scott W. et al.Predicting bioprocess targets of chemical compounds through integration of chemical-genetic and genetic interactionsPLoS Computational Biology2018ISSN 1553-7358
Nixon, Gemma L. et al.Repurposing and reformulation of the antiparasitic agent flubendazole for treatment of cryptococcal meningoencephalitis, a neglected fungal diseaseAntimicrobial Agents and Chemotherapy2018ISSN 1098-6596
Sato-Kaneko, Fumi et al.Discovery of a Novel Microtubule Targeting Agent as an Adjuvant for Cancer ImmunotherapyBioMed Research International2018ISSN 2314-6141
Dilworth, David et al.The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stabilityNucleic Acids Research2018ISSN 1362-4962
Huan, L C et al.Exploration of Some Thiazolidine-2, 4-dione and 2-Oxoindoline Derivatives Incorporating 3, 4, 5-Trimethoxybenzyl Moiety as Novel Anticancer AgentsLetters in Drug …2018Article Link
Qi, Jianguo et al.Synthesis and biological evaluation of N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxylic acid derivatives as tubulin polymerization inhibitorsEuropean Journal of Medicinal Chemistry2018ISSN 1768-3254
Senese, Silvia et al.Microtubins: A novel class of small synthetic microtubule targeting drugs that inhibit cancer cell proliferationOncotarget2017ISSN 1949-2553
Wieczorek, Anna et al.Synthesis and evaluation of biological properties of ferrocenyl-podophyllotoxin conjugatesDalton Transactions2017ISSN 1477-9234
Sun, Maolin et al.Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors with potent antiproliferative activityPLoS ONE2017ISSN 1932-6203
Zollo, Massimo et al.PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairmentBrain2017ISSN 1460-2156
Chen, Minfeng et al.Pericyte-targeting prodrug overcomes tumor resistance to vascular disrupting agentsJournal of Clinical Investigation2017ISSN 1558-8238
Tantak, Mukund P. et al.Design and synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel tubulin inhibitorsEuropean Journal of Medicinal Chemistry2017ISSN 1768-3254
Xu, Qile et al.Design, synthesis and structure-Activity relationship of 3,6-diaryl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines as novel tubulin inhibitorsScientific Reports2017ISSN 2045-2322
Kowalczyk, Karolina et al.Colchicine metallocenyl bioconjugates showing high antiproliferative activities against cancer cell linesDalton Transactions2017ISSN 1477-9234
Wu, Yue et al.Design and synthesis of 5-aryl-4-(4-arylpiperazine-1-carbonyl)-2H-1,2,3-triazole derivatives as colchicine binding site inhibitorsScientific Reports2017ISSN 2045-2322
Fu, Ying et al.The contrasting catalytic efficiency and cancer cell antiproliferative activity of stereoselective organoruthenium transfer hydrogenation catalystsDalton Transactions2016ISSN 1477-9234
Cai, De et al.YSL-12, a novel microtubule-destabilizing agent, exerts potent anti-tumor activity against colon cancer in vitro and in vivoCancer Chemotherapy and Pharmacology2016ISSN 1432-0843
Magalhaes, Luma G. et al.Discovery of a series of acridinones as mechanism-based tubulin assembly inhibitors with anticancer activityPLoS ONE2016ISSN 1932-6203
Hasanpourghadi, Mohadeseh et al.Targeting of tubulin polymerization and induction of mitotic blockage by Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (MBIC) in human cervical cancer HeLa cellJournal of Experimental and Clinical Cancer Research2016ISSN 1756-9966
Pearson, Brandon L. et al.Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegenerationNature Communications2016ISSN 2041-1723
Yoshitake, Jun et al.Modification of tau by 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP): Effects of nitric oxide-linked chemical modification on tau aggregationJournal of Biological Chemistry2016ISSN 1083-351X
Thapa, Pritam et al.Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel ChemotherapyJournal of Medicinal Chemistry2016ISSN 1520-4804
Fukuda, Yasunori et al.Tubulin is a molecular target of the Wnt-activating chemical probeBMC Biochemistry2016ISSN 1471-2091
Kamal, Ahmed et al.Design, synthesis and antiproliferative activity of the new conjugates of E7010 and resveratrol as tubulin polymerization inhibitorsOrganic and Biomolecular Chemistry2016ISSN 1477-0520
Fawzy, Iten M. et al.Newly Designed and Synthesized Curcumin Analogs with in vitro Cytotoxicity and Tubulin Polymerization ActivityChemical biology & drug design2015ISSN 1747--0285
Mu, Yan et al.The novel tubulin polymerization inhibitor MHPT exhibits selective anti-tumor activity against rhabdomyosarcoma in vitro and in vivoPLoS ONE2015ISSN 1932-6203
Xu, Qile et al.Synthesis and biological evaluation of 3-alkyl-1,5-diaryl-1H-pyrazoles as rigid analogues of combretastatin A-4 with potent antiproliferative activityPLoS ONE2015ISSN 1932-6203
Seashore-Ludlow, Brinton et al.Harnessing connectivity in a large-scale small-molecule sensitivity datasetCancer Discovery2015ISSN 2159-8290
Remers, William A. et al.Synthesis and Antitumor Activity of Heterocycles Related to CarbendazimJournal of Heterocyclic Chemistry2015ISSN 1943--5193
Sheldon, Jonathon E. et al.Photoswitchable anticancer activity via trans-cis isomerization of a combretastatin A-4 analogOrganic and Biomolecular Chemistry2015ISSN 1477-0520
Calles, Antonio et al.Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non-small-cell lung cancer cell linesMolecular Oncology2015ISSN 1878-0261
Yan, Jun et al.A novel synthetic compound exerts effective anti-tumour activity in vivo via the inhibition of tubulin polymerisation in A549 cellsBiochemical Pharmacology2015ISSN 1873-2968
Wu, Shaoyu et al.Bis-cyclopropane analog of disorazole C1 is a microtubuledestabilizing agent active in ABCB1-overexpressing human colon cancer cellsOncotarget2015ISSN 1949-2553
Shigehiro, Tsukasa et al.Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes PreparationPLOS ONE2014ISSN 1932--6203
Choi, Bo Hwa et al.Suprafenacine, an Indazole-Hydrazide Agent, Targets Cancer Cells Through Microtubule DestabilizationPLOS ONE2014ISSN 1932--6203
Senese, S. et al.Chemical dissection of the cell cycle: Probes for cell biology and anti-cancer drug developmentCell Death and Disease2014ISSN 2041-4889
Lutz, Vanessa et al.SAR studies on hydropentalene derivatives - Important core units of biologically active tetramic acid macrolactams and ptychanolidesBioorganic and Medicinal Chemistry2014ISSN 1464-3391
Mei, Mei et al.A new 2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene (SIA) derivative overcomes paclitaxel resistance by inhibiting MAPK signaling and increasing paclitaxel accumulation in breast cancer cellsPLoS ONE2014ISSN 1932-6203
Bio, Moses et al.Far-red light activatable, multifunctional prodrug for fluorescence optical imaging and combinational treatmentJournal of Medicinal Chemistry2014ISSN 1520-4804
Mäki-Jouppila, Jenni H.E. et al.Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.NaMolecular Cancer Therapeutics2014ISSN 1538-8514
Zhang, Qiu et al.Anti-tumor selectivity of a novel Tubulin and HSP90 dual-targeting inhibitor in non-small cell lung cancer modelsBiochemical Pharmacology2013ISSN 1873-2968
Bio, Moses et al.Site-specific and far-red-light-activatable prodrug of combretastatin A-4 using photo-unclick chemistryJournal of Medicinal Chemistry2013ISSN 0022-2623
Zach, Frank et al.The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule associationHuman molecular genetics2012ISSN 1460--2083
Sidhaye, Venkataramana K. et al.A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and StabilityPLOS ONE2012ISSN 1932--6203
Nguyen, Tam Luong et al.Evading Pgp activity in drug-resistant cancer cells: A structural and functional study of antitubulin furan metotica compoundsMolecular Cancer Therapeutics2012ISSN 1535-7163
Fitzgerald, Daniel P. et al.TPI-287, a new taxane family member, reduces the brain metastatic colonization of breast cancer cellsMolecular Cancer Therapeutics2012ISSN 1535-7163
Kawaratani, Yasuyuki et al.New microtubule polymerization inhibitors comprising a nitrooxymethylphenyl groupBioorganic and Medicinal Chemistry2011ISSN 0968-0896
Dyrager, Christine et al.Inhibitors and promoters of tubulin polymerization: synthesis and biological evaluation of chalcones and related dienones as potential anticancer agentsBioorganic & medicinal chemistry2011ISSN 1464--3391
Hwang, Ji Hwan et al.Induction of tubulin polymerization and apoptosis in malignant mesothelioma cells by a new compound JBIR-23Cancer letters2011ISSN 1872--7980
Kim, Insook et al.Zinc stimulates tau S214 phosphorylation by the activation of Raf/mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase pathwayNeuroReport2011ISSN 0959-4965
Oussenko, Irina A. et al.Effect of on 01910.Na, an anticancer mitotic inhibitor, on cell-cycle progression correlates with RanGAP1 hyperphosphorylationCancer Research2011ISSN 0008-5472

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