Tubulin protein (biotin): porcine brain

Tubulin protein (biotin): porcine brain
$0.00

Product Uses Include

  • Immobilizing tubulin onto a solid surface via streptavidin.
  • Creating tubulin or microtubule affinity matrices with streptavidin coated sepharose beads
  • High through-put screening using a proximity assay.
  • Microinjection into cells followed by electron microscopy of streptavidin conjugated gold particles to determine the cellular localization of the tubulin.
  • Nanotechnology

Material
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified so that random surface lysines contain a covalently linked, long-chain biotin derivative. A long-chain biotin derivative was selected for this procedure because it allows the biotin molecules to be spaced far enough away from the tubulin protein so as not to interfere with its activity, e.g., ligand binding to SPA beads or other streptavidin based reagents. Biotin labeled tubulin is supplied as a lyophilized powder.

Cytoskeleton, Inc. also offers biotinylated cancer cell tubulin (Cat. # H003 )

Purity
The protein purity of the tubulin used for labeling is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. The protein used for T333P is >99% pure tubulin. Labeled protein is run on an SDS gel, transfered to a nitrocellulose membrane and detected with streptavidin alkaline phosphatase (Fig 1). 10 ng of T333P is readily detectable. No free biotin is detectable in the final product.

t333blot

Figure 1: Purity determination of biotin tubulin. 10 and 100 ng of T333P was run on a 4-20% SDS-PAGE gel, transferred to a nitrocellulose membrane and biotin labeled material was detected with streptavidin alkaline phosphatase.

Biological Activity
The biological activity of T333P is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of fluorescein labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.

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

Havelka, D., Zhernov, I., Teplan, M. et al. Lab-on-chip microscope platform for electro-manipulation of a dense microtubules network. Sci Rep 12, 2462 (2022). https://doi.org/10.1038/s41598-022-06255-y (2022)

Peña, A. et al. Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy. Structure 28, 450-457.e5 (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).

Chudinova, E. M., Brodsky, I. B. & Nadezhdina, E. S. On the interaction of ribosomal protein RPL22e with microtubules. Cell Biol. Int. 43, 749–759 (2019).

Faltova, L. et al. Crystal Structure of a Heterotetrameric Katanin p60:p80 Complex. Structure 27, 1375-1383.e3 (2019).

Kim, E. et al. Electrical control of kinesin{\textendash}microtubule motility using a transparent functionalized-graphene substrate. Nanotechnology 24, 195102 (2013).

DeBerg, Hannah A et al. “Motor domain phosphorylation modulates kinesin-1 transport.” The Journal of biological chemistry vol. 288,45 (2013): 32612-21. doi:10.1074/jbc.M113.515510

B.-H. Choi et al. 2014. Suprafenacine, an indazole-hydrazide agent, targets cancer cells through microtubule destabilization. PLoS ONE. 9, e110955.

A.T. Lam et al. 2014. Controlling self-assembly of microtubule spools via kinesin motor density.  Soft Matter. 10, 8731-8736.

Dixit and Ross 2010. Chapter 27-Studying plus-end tracking at single molecule resolution using TIRF microscopy. Methods in Cell Biology. 95, 543-554.

Gell et al. 2010. Chapter 13-Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods in Cell Biology. 95, 221-245.

AuthorTitleJournalYearArticle Link
Zheng, Pengli et al.ER proteins decipher the tubulin code to regulate organelle distributionNature2022ISSN 1476-4687
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
Havelka, Daniel et al.Lab-on-chip microscope platform for electro-manipulation of a dense microtubules networkScientific Reports2022ISSN 2045-2322
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
Chen, Jiayi et al.Α-Tubulin Tail Modifications Regulate Microtubule Stability Through Selective Effector Recruitment, Not Changes in Intrinsic Polymer DynamicsDevelopmental Cell2021ISSN 1878-1551
Watson, Joseph L. et al.High-efficacy subcellular micropatterning of proteins using fibrinogen anchorsJournal of Cell Biology2021ISSN 1540-8140
Zehr, Elena A. et al.Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever MicrotubulesDevelopmental Cell2020ISSN 1878-1551
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020ISSN 1879-0445
Peña, Alejandro et al.Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron MicroscopyStructure2020ISSN 1878-4186
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
Zhernov, Ilia et al.Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional DiversityCurrent Biology2020ISSN 1879-0445
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020ISSN 1879-0445
Chudinova, Elena M. et al.On the interaction of ribosomal protein RPL22e with microtubulesCell Biology International2019ISSN 1095-8355
Li, Feiran et al.Local direction change of surface gliding microtubulesBiotechnology and Bioengineering2019ISSN 1097-0290
Nakos, Konstantinos et al.Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- And filament-dependent mannerMolecular Biology of the Cell2019ISSN 1939-4586
Faltova, Lenka et al.Crystal Structure of a Heterotetrameric Katanin p60:p80 ComplexStructure2019ISSN 1878-4186
Nakos, Konstantinos et al.Regulation of microtubule plus end dynamics by septin 9Cytoskeleton2019ISSN 1949-3592
Hu, Yuhan et al.Regulation of MT dynamics via direct binding of an Abl family kinaseJournal of Cell Biology2019ISSN 1540-8140
Siddiqui, Nida et al.PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular transportNature Communications2019ISSN 2041-1723
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
Hilton, Nicholas A. et al.Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesisMolecular Microbiology2018ISSN 1365-2958
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
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
Fan, Yuanwei et al.The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus EndsCurrent Biology2018ISSN 0960-9822
Pesenti, Marion E. et al.Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80Molecular Cell2018ISSN 1097-4164
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
McIntosh, Betsy B. et al.Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal NetworksCurrent Biology2018ISSN 0960-9822
Rao, Lu et al.Combining structure–function and single-molecule studies on cytoplasmic dyneinMethods in Molecular Biology2018ISSN 1064-3745
Reinemann, Dana N. et al.Processive Kinesin-14 HSET Exhibits Directional Flexibility Depending on Motor TrafficCurrent Biology2018ISSN 0960-9822
McClintock, Mark A. et al.RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPseLife2018ISSN 2050-084X
Aher, Amol et al.CLASP Suppresses Microtubule Catastrophes through a Single TOG DomainDevelopmental Cell2018ISSN 1878-1551
Zhang, Kai et al.Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and ActivatedCell2017ISSN 1097-4172
Arellano-Santoyo, Hugo et al.A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase ActivityDevelopmental Cell2017ISSN 1878-1551
Kandel, Mikhail E. et al.Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference MicroscopyACS Nano2017ISSN 1936-086X
Maciejowski, John et al.Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome SegregationDevelopmental Cell2017ISSN 1878-1551
Reinemann, Dana N. et al.Collective Force Regulation in Anti-parallel Microtubule Gliding by Dimeric Kif15 Kinesin MotorsCurrent Biology2017ISSN 0960-9822
Britto, Mishan et al.Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanismProceedings of the National Academy of Sciences of the United States of America2016ISSN 1091-6490
Szyk, Agnieszka et al.Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferaseCell2014ISSN 1097-4172
Lam, A. T. et al.Controlling self-assembly of microtubule spools via kinesin motor densitySoft Matter2014ISSN 1744-6848
Taberner, Núria et al.Reconstituting Functional Microtubule-Barrier InteractionsMethods in Cell Biology2014ISSN 0091-679X
Makrantoni, Vasso et al.Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiaePLoS ONE2014ISSN 1932-6203
Roth-Johnson, Elizabeth A. et al.Interaction between microtubules and the drosophila formin cappuccino and its effect on actin assemblyJournal of Biological Chemistry2014ISSN 0021-9258
Kim, Eunji et al.Electrical control of kinesin-microtubule motility using a transparent functionalized-graphene substrateNanotechnology2013ISSN 0957-4484
Yang, Yali et al.Microrheology of highly crosslinked microtubule networks is dominated by force-induced crosslinker unbindingSoft Matter2013ISSN 1744-683X
DeBerg, Hannah A. et al.Motor domain phosphorylation modulates kinesin-1 transportJournal of Biological Chemistry2013ISSN 0021-9258
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
Leslie, Kris et al.Going Solo: Measuring the motions of microtubules with an in vitro assay for tirf microscopy.Methods in Cell Biology2013ISSN 0091-679X
He, Siheng et al.Modeling negative cooperativity in streptavidin adsorption onto biotinylated microtubulesLangmuir2012ISSN 0743-7463
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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:  Does the biotinylated tubulin polymerize as well as unlabeled tubulin?

Answer 2:  Yes,  the biotinylated tubulin (Cat. # T333P) polymerizes as well as unlabeled tubulin (Cat. # 240).  The biological activity of biotinylated tubulin is determined from its ability to efficiently polymerize into microtubules in vitro and separate from unpolymerized protein in a spin-down assay.  Stringent quality control ensures that >85% of the biotinylated tubulin can polymerize in this assay.  This is comparable to the polymerization capacity of unmodified tubulin (Cat. # T240).

 

Question 3:  Why does Cytoskeleton recommend the use of general tubulin buffer and GTP for resuspending tubulin?

Answer 3:  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.