Tubulin protein (>99% pure): porcine brain

Tubulin protein (>99% pure): porcine brain
$0.00

Product Uses Include

  • IC50 & EC50 determinations for anti-tubulin ligands.
  • Microtubule binding studies
  • Tubulin monomer binding studies
  • HDAC6 studies
  • Microtubule activated kinesin ATPase assays

Material
Tubulin protein has been purified from porcine brain by an adaptation of the method of Shelanski et al. (1), Further purification to >99% purity was achieved by cation exchange chromatography. Tubulin is supplied as a white lyophilized powder.

Fully active for polymerization, this product is lyophilized with a patented technology for increased stability and longevity. T240 is stable for 1 year at 4°C desiccated. If your project requires the same batch of tubulin for consistent results, it is highly recommended that the item is purchased in bulk in order to save time and money. This product can be used as a substitute for our highly purified bovine tubulin products (Cat. # TL238, T238 and T237) and behaves in an identical fashion.

Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >99% pure

t240

Figure 1: A 20 µg sample of T240 protein was separated by electrophoresis on a 10% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02).

Biological Activity
One unit of tubulin is defined as 5.0 mg of purified protein (as determined by the Precision Red Advanced Protein Assay Reagent cat. # ADV02). The biological activity of T240 is assessed by a tubulin polymerization assay. The ability of tubulin to polymerize into microtubules can be followed by observing an increase in optical density of the tubulin solution at 340 nm. A 5 mg/ml tubulin solution in General Tubulin Buffer buffer plus 5% glycerol and 1 mM GTP should achieve an OD340 nm reading between 0.75-1.10 in 30 min at 37°C when using a spectrophotometer pathlength of 0.8 cm (180 µl sample volume in a 1/2 area 96-well plate).

It should be noted that tubulin minus glycerol WILL NOT polymerize in G-PEM buffer until very high tubulin concentrations (>10 mg/ml). Even at these concentrations polymerization is comparatively slow. Efficient polymerization at low concentration of tubulin minus glycerol can be achieved by addition of a polymerization stimulating compound, e.g., glycerol, paclitaxel or DMSO.

References

Shelanski, M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768

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

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Farhadi, Leila et al. “Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network.” Soft matter vol. 16,31 (2020): 7191-7201. doi:10.1039/c9sm02400j (2021)

Ricketts, S. N. et al. Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics. Front. Phys. 8, 494 (2020).

Alatrash, N. et al. Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex. Chem. Sci. 11, 264–275 (2020).

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 Kalra, Aarat P. et al. “Investigation of the Electrical Properties of Microtubule Ensembles Under Cell-Like Conditions.” Nanomaterials 10.2 (2020): 265.

Kaur, S. et al. Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A). Human Mutation. 2020; 41: 1761– 1774. https://doi.org/10.1002/humu.24079

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

Hosono et al., 2012. The murine Gcap14 gene encodes a novel microtubule binding and bundling protein. FEBS Lett. v 586, pp 1426-1430.

Kawakami et al., 2012. LRRK2 Phosphorylates Tubulin-Associated Tau but Not the Free Molecule: LRRK2-Mediated Regulation of the Tau-Tubulin Association and Neurite Outgrowth. PLoS ONE. 7: e30834.

Berezniuk et al., 2012. Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin. J. Biol. Chem. 287, 6503-6517.

Wu et al., 2012. A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice. FASEB J. doi: 10.1096/fj.12-205047.

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Faivre-Moskalenko and Dogterom, 2002. Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes. Proc. Natl. Acad. Sci. U.S.A. v 99, pp 16788-16793.

AuthorTitleJournalYearArticle Link
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Yang, Yun et al.Altered succinylation of mitochondrial proteins, APP and tau in Alzheimer’s diseaseNature Communications2022ISSN 2041-1723
Morris, Joseph D. et al.Re-evaluation of the Fijianolide/Laulimalide Chemotype Suggests an Alternate Mechanism of Action for C-15/C-20 AnalogsACS Omega2022ISSN 2470-1343
Capizzi, Mariacristina et al.Developmental defects in Huntington's disease show that axonal growth and microtubule reorganization require NUMA1Neuron2022ISSN 1097-4199
Bär, Julia et al.Regulation of microtubule detyrosination by calcium and conventional calpainsJournal of Cell …2022Article Link
Kuzmić, Mira et al.Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibersJournal of Cell Science2022ISSN 1477-9137
Baron, Desiree M et al.ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function.Cell reports2022ISSN 2211--1247
Feizabadi, Mitra Shojania et al.The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In VitroInternational Journal of Molecular Sciences2022ISSN 1422-0067
Budaitis, Breane G. et al.Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanismsJournal of Cell Biology2021ISSN 1540-8140
Cook, Alexander D. et al.Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibitionJournal of Biological Chemistry2021ISSN 1083-351X
Yu, Xian et al.MARK4 controls ischaemic heart failure through microtubule detyrosinationNature2021ISSN 1476-4687
Alfieri, Angus et al.Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruptionCurrent Biology2021ISSN 1879-0445
Tang, Qing et al.NDST3 deacetylates α‐tubulin and suppresses V‐ATPase assembly and lysosomal acidificationThe EMBO Journal2021ISSN 0261--4189
Yano, Tomoki et al.A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constrictionThe EMBO Journal2021ISSN 0261--4189
Nourbakhsh, Kimya et al.TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubulesDevelopmental Cell2021ISSN 1878-1551
Kundu, Tanushree et al.Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth conesJournal of Cell Science2021ISSN 1477-9137
Ludzia, Patryk et al.Structural characterization of KKT4, an unconventional microtubule-binding kinetochore proteinStructure2021ISSN 1878-4186
Deshpande, Ojas et al.Astral microtubule cross-linking safeguards uniform nuclear distribution in the drosophila syncytiumJournal of Cell Biology2021ISSN 1540-8140
Nakayama, Shogo et al.Planar cell polarity induces local microtubule bundling for coordinated ciliary beatingJournal of Cell Biology2021ISSN 1540-8140
Habicht, Juri et al.UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin IIJournal of Cell Science2021ISSN 1477-9137
Gao, Li et al.A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule CytoskeletonCell Chemical Biology2021ISSN 2451-9448
Watson, Joseph L. et al.High-efficacy subcellular micropatterning of proteins using fibrinogen anchorsJournal of Cell Biology2021ISSN 1540-8140
Peña, Alejandro et al.Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron MicroscopyStructure2020ISSN 1878-4186
Munari, Francesca et al.Semisynthetic modification of tau protein with di-ubiquitin chains for aggregation studiesInternational Journal of Molecular Sciences2020ISSN 1422-0067
Müller-Deku, Adrian et al.Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeletonNature Communications2020ISSN 2041-1723
Chen, Keyu et al.Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubulesJournal of Cell Biology2020ISSN 1540-8140
Diwaker, Drishya et al.Deletion of the Pseudorabies virus gE/gI-US9p complex disrupts kinesin KIF1A and KIF5C recruitment during egress, and alters the properties of microtubule-dependent transport in vitroPLoS Pathogens2020ISSN 1553-7374
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020ISSN 1879-0445
Atherton, Joseph et al.The mechanism of kinesin inhibition by kinesin binding proteineLife2020ISSN 2050-084X
Saper, Gadiel et al.Kinesin-propelled label-free microtubules imaged with interference reflection microscopyNew Journal of Physics2020ISSN 1367-2630
Martinez, Pablo et al.TANGLED1 mediates microtubule interactions that may promote division plane positioning in maizeJournal of Cell Biology2020ISSN 1540-8140
Adriaans, Ingrid E. et al.MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during AnaphaseCurrent Biology2020ISSN 1879-0445
Gaska, Ignas et al.The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule SlidingDevelopmental Cell2020ISSN 1878-1551
Steib, Emmanuelle et al.Wdr90 is a centriolar microtubule wall protein important for centriole architecture integrityeLife2020ISSN 2050-084X
Gunji, Shizuka et al.Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thalianaFrontiers in Plant Science2020ISSN 1664-462X
Kraus, Yvonne et al.Isoquinoline-based biaryls as a robust scaffold for microtubule inhibitorsEuropean Journal of Medicinal Chemistry2020ISSN 1768-3254
Alatrash, Nagham et al.Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complexChemical Science2020ISSN 2041-6539
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020ISSN 1879-0445
Mahalingan, Kishore K. et al.Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymesNature Structural and Molecular Biology2020ISSN 1545-9985
Ouyang, Changhan et al.Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylationAutophagy2020ISSN 1554-8635
Zhang, Shengnan et al.In-cell NMR study of Tau and MARK2 phosphorylated TauInternational Journal of Molecular Sciences2019ISSN 1422-0067
Grueb, Saskia S. et al.The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domainScientific Reports2019ISSN 2045-2322
Leong, Su Ling et al.Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1Current Biology2019ISSN 0960-9822
Ng, Cai Tong et al.Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situJournal of Cell Biology2019ISSN 1540-8140
Jiang, Shuo et al.Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7Developmental Cell2019ISSN 1878-1551
Xie, Shuwei et al.MICAL-L1 coordinates ciliogenesis by recruiting EHD1 to the primary ciliumJournal of Cell Science2019ISSN 1477-9137
Nakos, Konstantinos et al.Regulation of microtubule plus end dynamics by septin 9Cytoskeleton2019ISSN 1949-3592
Lopes, Joseph et al.Membrane mediated motor kinetics in microtubule gliding assaysScientific Reports2019ISSN 2045-2322
Faltova, Lenka et al.Crystal Structure of a Heterotetrameric Katanin p60:p80 ComplexStructure2019ISSN 1878-4186
Guzman-Sepulveda, Jose Rafael et al.Tubulin Polarizability in Aqueous SuspensionsACS Omega2019ISSN 2470-1343
Aher, Amol et al.CLASP Suppresses Microtubule Catastrophes through a Single TOG DomainDevelopmental Cell2018ISSN 1878-1551
Kong, Ji Na et al.Novel function of ceramide for regulation of mitochondrial ATP release in astrocytesJournal of Lipid Research2018ISSN 1539-7262
Revenkova, Ekaterina et al.The Joubert syndrome protein ARL13B binds tubulin to maintain uniform distribution of proteins along the ciliary membraneJournal of Cell Science2018ISSN 1477-9137
Parida, Pravat Kumar et al.Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosisCell Death and Disease2018ISSN 2041-4889
Karasmanis, Eva P. et al.Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))Developmental Cell2018ISSN 1878-1551
Tu, Hai Qing et al.Microtubule asters anchored by FSD1 control axoneme assembly and ciliogenesisNature Communications2018ISSN 2041-1723
Howes, Stuart C. et al.Structural and functional differences between porcine brain and budding yeast microtubulesCell Cycle2018ISSN 1551-4005
Karasmanis, Eva P. et al.Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))Developmental Cell2018ISSN 1878-1551
McClintock, Mark A. et al.RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPseLife2018ISSN 2050-084X
Karasmanis, Eva P. et al.Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))Developmental Cell2018ISSN 1878-1551
Ganguly, Anindya et al.Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4Developmental Cell2018ISSN 1878-1551
Rao, Lu et al.Combining structure–function and single-molecule studies on cytoplasmic dyneinMethods in Molecular Biology2018ISSN 1064-3745
Fan, Yuanwei et al.The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus EndsCurrent Biology2018ISSN 0960-9822
Romé, Pierre et al.A novel microtubule nucleation pathway for meiotic spindle assembly in oocytesJournal of Cell Biology2018ISSN 1540-8140
Tripathy, Ratna et al.Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical MalformationsNeuron2018ISSN 1097-4199
Karasmanis, Eva P. et al.Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))Developmental Cell2018ISSN 1878-1551
Schellhaus, Anna Katharina et al.Developmentally Regulated GTP binding protein 1 (DRG1) controls microtubule dynamicsScientific Reports2017ISSN 2045-2322
Kandel, Mikhail E. et al.Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference MicroscopyACS Nano2017ISSN 1936-086X
Okeyoshi, Kosuke et al.Methods for the self-integration of megamolecular biopolymers on the drying air-LC interfaceJournal of Visualized Experiments2017ISSN 1940-087X
Majellaro, Maria et al.Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl NicotinamidesChemMedChem2017ISSN 1860-7187
Onder, Seda et al.Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino AcidsChemical Research in Toxicology2017ISSN 1520-5010
Monda, Julie K. et al.Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting SurfacesCurrent Biology2017ISSN 0960-9822
Lindamulage, I. Kalhari et al.Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 functionScientific Reports2017ISSN 2045-2322
Howes, Stuart C. et al.Structural differences between yeast and mammalian microtubules revealed by cryo-EMJournal of Cell Biology2017ISSN 1540-8140
Gramlich, Michael W. et al.Single molecule investigation of kinesin-1 motility using engineered microtubule defectsScientific Reports2017ISSN 2045-2322
Murray, John W. et al.Reduction of organelle motility by removal of potassium and other solutesPLoS ONE2017ISSN 1932-6203
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Ayoub, Ahmed Taha et al.Antitumor Activity of Lankacidin Group Antibiotics Is Due to Microtubule Stabilization via a Paclitaxel-like MechanismJournal of Medicinal Chemistry2016ISSN 1520-4804
Bartsch, Tobias F. et al.Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solutionNature Communications2016ISSN 2041-1723
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Question 1:  What is the proper way to store the tubulin to insure maximum stability and activity?

Answer 1:  The recommended storage condition for the lyophilized tubulin product is 4°C with desiccant to maintain humidity at <10% humidity.  Under these conditions the protein is stable for 6 months.  Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months.  However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture.  Therefore we recommend storing at 4°C.  If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized.  After reconstituting the protein as directed, the concentrated protein in G-PEM buffer should be aliquoted, snap frozen in liquid nitrogen and stored at -70°C (stable for 6 months).  NOTE: It is very important to snap freeze the tubulin in liquid nitrogen as other methods of freezing will result in significantly reduced activity.  Defrost rapidly by placing in a room temperature water bath for 1 min.  Avoid repeated freeze/thaw cycles.

 

Question 2:  Why does Cytoskeleton recommend the use of general tubulin buffer and GTP for resuspending tubulin?

Answer 2:  We recommend resuspending tubulin in general tubulin buffer + GTP to maintain tubulin monomer protein stability and conformation and to provide the necessary components for polymerization.  For resuspension, we recommend using a general tubulin buffer (Cat. # BST01-001) which consists of 80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 7.0, supplemented with 1 mM GTP (Cat. # BST06-001).  Tubulin requires GTP and magnesium ions for proper stability and conformation, even in its monomeric state.  GTP is also required for the polymerization process as its hydrolysis during tubulin polymerization is necessary for polymerization to occur.  EGTA is a chelator of calcium which is a potent inhibitor of tubulin polymerization.  Glycerol is often added to a final concentration of 5 - 10% to enhance polymerization; however, glycerol is not necessary for the maintenance of biologically active tubulin and does not need to be included when reconstituting and storing tubulin.  When aliquoting reconstituted tubulin for storage, it is essential to aliquot and snap-freeze tubulin in liquid nitrogen at a concentration of >6 mg/ml to preserve tubulin’s biological activity.  Then the aliquots should be stored at -70°C.  When thawing the aliquots, thaw rapidly in a room temperature water bath and place on ice until right before experimental use.

 

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