Tubulin protein (>99% pure): porcine brain

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T240

Datasheet

MSDS

Amount

1x1 mg (Cat. # T240-A) + $129.00

5x1 mg (Cat. # T240-B) + $582.00

20x1 mg (Cat. # T240-C) + $1,906.00

10x1 mg (Cat. # T240-D) + $1,020.00

1x10 mg (Cat. # T240-DX) + $912.00

Qty:

Details

Product Uses Include

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

Click Here for Detailed Methods

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

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 OD_{340 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

About

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

Citations

Author	Title	Journal	Year	Article Link
Henty-Ridilla, Jessica L.	Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy	JoVE (Journal of Visualized Experiments)	2022	ISSN 1940--087X
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Baron, Desiree M et al.	ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function.	Cell reports	2022	ISSN 2211--1247
Morris, Joseph D. et al.	Re-evaluation of the Fijianolide/Laulimalide Chemotype Suggests an Alternate Mechanism of Action for C-15/C-20 Analogs	ACS Omega	2022	ISSN 2470-1343
Wu, R. et al.	Thermal hysteresis in microtubule assembly/disassembly dynamics: The aging-induced degradation of tubulin dimers	Biochemistry and Biophysics Reports	2022	ISSN 2405-5808
Planelles-Herrero, Vicente Jose et al.	Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants	Nature Cell Biology	2022	ISSN 1476--4679
Capizzi, Mariacristina et al.	Developmental defects in Huntington's disease show that axonal growth and microtubule reorganization require NUMA1	Neuron	2022	ISSN 1097-4199
Kuzmić, Mira et al.	Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers	Journal of Cell Science	2022	ISSN 1477-9137
Yang, Yun et al.	Altered succinylation of mitochondrial proteins, APP and tau in Alzheimer’s disease	Nature Communications	2022	ISSN 2041-1723
Bär, Julia et al.	Regulation of microtubule detyrosination by calcium and conventional calpains	Journal of Cell …	2022	Article Link
Hough, Cameron M. et al.	Disassembly of microtubules by intense terahertz pulses	Biomedical Optics Express	2021	ISSN 2156--7085
Budaitis, Breane G. et al.	Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanisms	Journal of Cell Biology	2021	ISSN 1540-8140
Yano, Tomoki et al.	A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction	The EMBO Journal	2021	ISSN 0261--4189
Ludzia, Patryk et al.	Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein	Structure	2021	ISSN 1878-4186
Nakayama, Shogo et al.	Planar cell polarity induces local microtubule bundling for coordinated ciliary beating	Journal of Cell Biology	2021	ISSN 1540-8140
Tang, Qing et al.	NDST3 deacetylates α‐tubulin and suppresses V‐ATPase assembly and lysosomal acidification	The EMBO Journal	2021	ISSN 0261--4189
Yu, Xian et al.	MARK4 controls ischaemic heart failure through microtubule detyrosination	Nature	2021	ISSN 1476-4687
Gao, Li et al.	A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton	Cell Chemical Biology	2021	ISSN 2451-9448
Lee, Gloria et al.	Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics	Science Advances	2021	ISSN 2375-2548
Alfieri, Angus et al.	Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption	Current Biology	2021	ISSN 1879-0445
Habicht, Juri et al.	UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin II	Journal of Cell Science	2021	ISSN 1477-9137
Nourbakhsh, Kimya et al.	TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules	Developmental Cell	2021	ISSN 1878-1551
Kundu, Tanushree et al.	Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth cones	Journal of Cell Science	2021	ISSN 1477-9137
Watson, Joseph L. et al.	High-efficacy subcellular micropatterning of proteins using fibrinogen anchors	Journal of Cell Biology	2021	ISSN 1540-8140
Elahi, Montasir et al.	High-fat diet–induced activation of SGK1 promotes Alzheimer’s disease–associated tau pathology	Human Molecular Genetics	2021	ISSN 0964--6906
Zhou, Libin et al.	Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration	Scientific Reports 2021 11:1	2021	ISSN 2045--2322
Deshpande, Ojas et al.	Astral microtubule cross-linking safeguards uniform nuclear distribution in the drosophila syncytium	Journal of Cell Biology	2021	ISSN 1540-8140
Gaska, Ignas et al.	The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule Sliding	Developmental Cell	2020	ISSN 1878-1551
Atherton, Joseph et al.	The mechanism of kinesin inhibition by kinesin binding protein	eLife	2020	ISSN 2050-084X
Ricketts, Shea N. et al.	Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics	Frontiers in Physics	2020	ISSN 2296-424X
Gunji, Shizuka et al.	Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana	Frontiers in Plant Science	2020	ISSN 1664-462X
Aher, Amol et al.	CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation	Current Biology	2020	ISSN 1879-0445
Kraus, Yvonne et al.	Isoquinoline-based biaryls as a robust scaffold for microtubule inhibitors	European Journal of Medicinal Chemistry	2020	ISSN 1768-3254
Mahalingan, Kishore K. et al.	Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes	Nature Structural and Molecular Biology	2020	ISSN 1545-9985
Chen, Keyu et al.	Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules	Journal of Cell Biology	2020	ISSN 1540-8140
Rodríguez-García, Ruddi et al.	Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules	Current Biology	2020	ISSN 1879-0445
Alatrash, Nagham et al.	Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex	Chemical Science	2020	ISSN 2041-6539
Saper, Gadiel et al.	Kinesin-propelled label-free microtubules imaged with interference reflection microscopy	New Journal of Physics	2020	ISSN 1367-2630
Müller-Deku, Adrian et al.	Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton	Nature Communications	2020	ISSN 2041-1723
Ouyang, Changhan et al.	Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation	Autophagy	2020	ISSN 1554-8635
Steib, Emmanuelle et al.	Wdr90 is a centriolar microtubule wall protein important for centriole architecture integrity	eLife	2020	ISSN 2050-084X
Adriaans, Ingrid E. et al.	MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase	Current Biology	2020	ISSN 1879-0445
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 vitro	PLoS Pathogens	2020	ISSN 1553-7374
Kaur, Simranpreet et al.	Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A)	Human Mutation	2020	ISSN 1098-1004
Martinez, Pablo et al.	TANGLED1 mediates microtubule interactions that may promote division plane positioning in maize	Journal of Cell Biology	2020	ISSN 1540-8140
Baker, Stacey J. et al.	A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent	Molecular Cell	2020	ISSN 1097-4164
Zhang, Shengnan et al.	In-cell NMR study of Tau and MARK2 phosphorylated Tau	International Journal of Molecular Sciences	2019	ISSN 1422-0067
Leong, Su Ling et al.	Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1	Current Biology	2019	ISSN 0960-9822
Francis, Madison L. et al.	Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites	Soft Matter	2019	ISSN 1744-6848
Lopes, Joseph et al.	Membrane mediated motor kinetics in microtubule gliding assays	Scientific Reports	2019	ISSN 2045-2322
Nakos, Konstantinos et al.	Regulation of microtubule plus end dynamics by septin 9	Cytoskeleton	2019	ISSN 1949-3592
Jiang, Shuo et al.	Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7	Developmental Cell	2019	ISSN 1878-1551
Guzman-Sepulveda, Jose Rafael et al.	Tubulin Polarizability in Aqueous Suspensions	ACS Omega	2019	ISSN 2470-1343
Xie, Shuwei et al.	MICAL-L1 coordinates ciliogenesis by recruiting EHD1 to the primary cilium	Journal of Cell Science	2019	ISSN 1477-9137
Faltova, Lenka et al.	Crystal Structure of a Heterotetrameric Katanin p60:p80 Complex	Structure	2019	ISSN 1878-4186
Ng, Cai Tong et al.	Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ	Journal of Cell Biology	2019	ISSN 1540-8140
Aher, Amol et al.	CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain	Developmental Cell	2018	ISSN 1878-1551
Revenkova, Ekaterina et al.	The Joubert syndrome protein ARL13B binds tubulin to maintain uniform distribution of proteins along the ciliary membrane	Journal of Cell Science	2018	ISSN 1477-9137
Rao, Lu et al.	Combining structure–function and single-molecule studies on cytoplasmic dynein	Methods in Molecular Biology	2018	ISSN 1064-3745
McClintock, Mark A. et al.	RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs	eLife	2018	ISSN 2050-084X
Romé, Pierre et al.	A novel microtubule nucleation pathway for meiotic spindle assembly in oocytes	Journal of Cell Biology	2018	ISSN 1540-8140
Fan, Yuanwei et al.	The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus Ends	Current Biology	2018	ISSN 0960-9822
Ganguly, Anindya et al.	Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4	Developmental Cell	2018	ISSN 1878-1551
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 Cell	2018	ISSN 1878-1551
Tripathy, Ratna et al.	Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations	Neuron	2018	ISSN 1097-4199
Lindamulage, I. Kalhari et al.	Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function	Scientific Reports	2017	ISSN 2045-2322
Onder, Seda et al.	Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids	Chemical Research in Toxicology	2017	ISSN 1520-5010
Murray, John W. et al.	Reduction of organelle motility by removal of potassium and other solutes	PLoS ONE	2017	ISSN 1932-6203
Howes, Stuart C. et al.	Structural differences between yeast and mammalian microtubules revealed by cryo-EM	Journal of Cell Biology	2017	ISSN 1540-8140
Kandel, Mikhail E. et al.	Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy	ACS Nano	2017	ISSN 1936-086X
Okeyoshi, Kosuke et al.	Methods for the self-integration of megamolecular biopolymers on the drying air-LC interface	Journal of Visualized Experiments	2017	ISSN 1940-087X
Monda, Julie K. et al.	Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces	Current Biology	2017	ISSN 0960-9822
Majellaro, Maria et al.	Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl Nicotinamides	ChemMedChem	2017	ISSN 1860-7187
Gramlich, Michael W. et al.	Single molecule investigation of kinesin-1 motility using engineered microtubule defects	Scientific Reports	2017	ISSN 2045-2322
Ghaly, Peter E. et al.	A new antiproliferative noscapine analogue: Chemical synthesis and biological evaluation	Oncotarget	2016	ISSN 1949-2553
Lor, Chai et al.	A simple experimental model to investigate force range for membrane nanotube formation	Frontiers in Materials	2016	ISSN 2296-8016
Bartsch, Tobias F. et al.	Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solution	Nature Communications	2016	ISSN 2041-1723
Kellogg, Elizabeth H. et al.	Near-atomic cryo-EM structure of PRC1 bound to the microtubule	Proceedings of the National Academy of Sciences of the United States of America	2016	ISSN 1091-6490
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
Derbyshire, Paul et al.	Proteomic analysis of microtubule interacting proteins over the course of xylem tracheary element formation in Arabidopsis	Plant Cell	2015	ISSN 1532-298X
Hara, Yuki et al.	Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts	Developmental Cell	2015	ISSN 1878-1551
Matsuyama, Tomonori et al.	Midaz(olam) inhibits the hypoxia-induced up-regulation of erythropoietin in the central nervous system	European Journal of Pharmacology	2015	ISSN 1879-0712
Kim, Kyongwan et al.	Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodes	Journal of Applied Physics	2015	ISSN 1089-7550
Kim, Sehyun et al.	Nek2 activation of Kif24 ensures cilium disassembly during the cell cycle	Nature Communications	2015	ISSN 2041-1723
Roth-Johnson, Elizabeth A. et al.	Interaction between microtubules and the drosophila formin cappuccino and its effect on actin assembly	Journal of Biological Chemistry	2014	ISSN 0021-9258
Taberner, Núria et al.	Reconstituting Functional Microtubule-Barrier Interactions	Methods in Cell Biology	2014	ISSN 0091-679X
López, Magdalena Preciado et al.	Actin-microtubule coordination at growing microtubule ends	Nature Communications	2014	ISSN 2041-1723
Kim, Eunji et al.	Electrical control of kinesin-microtubule motility using a transparent functionalized-graphene substrate	Nanotechnology	2013	ISSN 0957-4484
Wu, Hui Yuan et al.	A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice	The FASEB Journal	2012	ISSN 1530-6860
Hosono, Hitomi et al.	The murine Gcap14 gene encodes a novel microtubule binding and bundling protein	FEBS letters	2012	ISSN 1873--3468
Berezniuk, Iryna et al.	Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin	The Journal of Biological Chemistry	2012	ISSN 0021-9258
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Kawakami, Fumitaka et al.	LRRK2 phosphorylates tubulin-associated tau but not the free molecule: LRRK2-mediated regulation of the tau-tubulin association and neurite outgrowth	PloS one	2012	ISSN 1932--6203
Kwok, Ester et al.	FES kinase promotes mast cell recruitment to mammary tumors via the stem cell factor/KIT receptor signaling axis	Molecular Cancer Research	2012	ISSN 1541-7786
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Faqs

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.

Question 3: How does porcine tubulin compare to bovine tubulin?

Answer 3: Click here for an in-depth comparison of porcine and bovine tubulin.

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