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Tubulin protein (rhodamine): porcine brain

Tubulin protein (rhodamine): porcine brain
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TL590M
Datasheet
MSDS
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Rhodamine labeled microtubules formed from rhodamine labeled tubulin.

TL590MMTs
TL590M_scan

Product Uses Include

  • Laser based applications
  • Monitoring microtubule dynamcs in living cells
  • Speckle microscopy
  • Formation of fluorescent microtubules
  • Microscopy studies of MAP and microtubule associated motor activities
  • Nanotechnology
Click Here for Detailed Methods

Material
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked rhodamine at random surface lysines. An activated ester of rhodamine was used to label the protein. Labeling stoichiometry was determined by spectroscopic measurement of protein and dye concentrations (dye extinction coefficient when protein bound is 64,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. rhodamine labeled tubulin can be detected using a filter set of 530-550 nm excitation and 580-600 emission. Rhodamine tubulin is in a versatile, stable and easily shipped format. It is ready for micro-injection or in vitro polymerization. Cytoskeleton, Inc. also offers AMCA (Cat. # TL440M), HiLyte Fluor™ 488 (Cat. # TL488M), X-rhodamine (Cat. # TL620M) and HiLyte Fluor™ 647TM (Cat. # TL670M) labeled tubulins of the same quality.

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 TL590M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated rhodamine dye would be visible in the dye front. No fluorescence is detected in the dye front, indicating that no free dye is present in the final product (Figure 1 B).

tl334mgels

Figure 1: Rhodamine tubulin protein purity determination. A 50 µg sample of unlabeled tubulin protein was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue (A). Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02). 20 µg of the same protein sample was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).

Biological Activity
The biological activity of rhodamine tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of rhodamine 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

AuthorTitleJournalYearArticle Link
Zhou, Qing et al.An orphan kinesin in Trypanosoma brucei regulates hook complex assembly and Golgi biogenesismBio2024
Sun, Shuangshuang et al.Metabolic regulation of cytoskeleton functions by HDAC6-catalyzed α-tubulin lactylationNature Communications 2024 15:12024
Lucas, Lathan et al.Alternatively Spliced MAP4 Isoforms Have Key Roles in Maintaining Microtubule Organization and Skeletal Muscle FunctioniScience2024
Queen, Katelyn A. et al.Modification of the neck-linker of KIF18A alters Microtubule subpopulation preferenceMolecular biology of the cell2024
Muhs, Stefanie et al.Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule DynamicsCells 2024
Luchniak, Anna et al.Tubulin CFEOM mutations both inhibit or activate kinesin motor activityMolecular Biology of the Cell2024
Fan, Yuanwei et al.A divergent tumor overexpressed gene domain and oligomerization contribute to SPIRAL2 function in stabilizing microtubule minus endsThe Plant Cell2024
Liu, Xinglei et al.Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokesNature Communications2024
Mahalingan, Kishore K. et al.Structural basis for α-tubulin-specific and modification state-dependent glutamylationNature Chemical Biology2024
Chauhan, Prashali et al.Ionic strength alters crosslinker-driven self-organization of microtubulesCytoskeleton2024
Jijumon, A. S. et al.A Platform for Medium-Throughput Cell-Free Analyses of Microtubule-Interacting Proteins Using Mammalian Cell LysatesCurrent Protocols2024
Hu, Huiqing et al.A kinesin-13 family kinesin in Trypanosoma brucei regulates cytokinesis and cytoskeleton morphogenesis by promoting microtubule bundlingPLOS Pathogens2024
Gluszek‐Kustusz, Agata et al. Phosphorylation controls spatial and temporal activities of motor‐PRC1 complexes to complete mitosis The EMBO Journal2023
Memarian, Fereshteh L. et al.Forming, Confining, and Observing Microtubule-Based Active NematicsJournal of Visualized Experiments (JoVE)2023
van den Berg, Cyntha M. et al.CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal sideJournal of Cell Biology2023
McGorty, Ryan J. et al.Kinesin and myosin motors compete to drive rich multiphase dynamics in programmable cytoskeletal compositesPNAS Nexus2023
Nithianantham, Stanley et al.The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activityMolecular biology of the cell2023
McHugh, Toni et al.Potent microtubule-depolymerizing activity of a mitotic Kif18b–MCAK–EB networkJournal of Cell Science2023
Yang, Shuzhen et al.EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesisNature Communications2023
Hoshino, Asumi et al.The microtubule-severing protein UNC-45A preferentially binds to curved microtubules and counteracts the microtubule-straightening effects of TaxolJournal of Biological Chemistry2023
Oda, Haruka et al.Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryoBiology Open2023
Palumbo, Jacob et al.Directly Measuring Forces Within Reconstituted Active Microtubule BundlesJoVE (Journal of Visualized Experiments)2022
Qian, Pengge et al.Apical anchorage and stabilization of subpellicular microtubules by apical polar ring ensures Plasmodium ookinete infection in mosquitoNature Communications 2022
Henty-Ridilla, Jessica L.Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) MicroscopyJoVE (Journal of Visualized Experiments)2022
Sasanpour, Mehrzad et al.Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and MechanicsJoVE (Journal of Visualized Experiments)2022
Planelles-Herrero, Vicente Jose et al.Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinantsNature Cell Biology 2022
Kuzmić, Mira et al.Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibersJournal of Cell Science2022
Lee, Gloria et al.Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamicsScience Advances2021
Hough, Cameron M. et al.Disassembly of microtubules by intense terahertz pulsesBiomedical Optics Express2021
Habicht, Juri et al.UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin IIJournal of Cell Science2021
Kundu, Tanushree et al.Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth conesJournal of Cell Science2021
Saper, Gadiel et al.Robotic end-to-end fusion of microtubules powered by kinesinScience Robotics2021
Alfieri, Angus et al.Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruptionCurrent Biology2021
Kaur, Simranpreet et al.Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A)Human Mutation2020
Ricketts, Shea N. et al.Triggering Cation-Induced Contraction of Cytoskeleton Networks via MicrofluidicsFrontiers in Physics2020
Pyrpassopoulos, Serapion et al.Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule TrackBiophysical Journal2020
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020
Chen, Keyu et al.Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubulesJournal of Cell Biology2020
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020
Saper, Gadiel et al.Kinesin-propelled label-free microtubules imaged with interference reflection microscopyNew Journal of Physics2020
Adriaans, Ingrid E. et al.MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during AnaphaseCurrent Biology2020
Gaska, Ignas et al.The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule SlidingDevelopmental Cell2020
Kalra, Aarat P. et al.Investigation of the electrical properties of microtubule ensembles under cell-like conditionsNanomaterials2020
Tuszynski, Jack A. et al.Microtubules as Sub-Cellular MemristorsScientific Reports2020
Francis, Madison L. et al.Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton compositesSoft Matter2019
Leong, Su Ling et al.Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1Current Biology2019
Lopes, Joseph et al.Membrane mediated motor kinetics in microtubule gliding assaysScientific Reports2019
Faltova, Lenka et al.Crystal Structure of a Heterotetrameric Katanin p60:p80 ComplexStructure2019
Grueb, Saskia S. et al.The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domainScientific Reports2019
Chudinova, Elena M. et al.On the interaction of ribosomal protein RPL22e with microtubulesCell Biology International2019
Fu, Meng meng et al.The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for MyelinationCell2019
Ricketts, Shea N. et al.Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule compositesScientific Reports2019
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 Cell2019
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 Cell2018
Ganguly, Anindya et al.Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4Developmental Cell2018
Fan, Yuanwei et al.The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus EndsCurrent Biology2018
Romé, Pierre et al.A novel microtubule nucleation pathway for meiotic spindle assembly in oocytesJournal of Cell Biology2018
Colin, Alexandra et al.Actin-Network Architecture Regulates Microtubule DynamicsCurrent Biology2018
Rao, Lu et al.Combining structure–function and single-molecule studies on cytoplasmic dyneinMethods in Molecular Biology2018
Aher, Amol et al.CLASP Suppresses Microtubule Catastrophes through a Single TOG DomainDevelopmental Cell2018
Hilton, Nicholas A. et al.Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesisMolecular Microbiology2018
Höing, Susanne et al.Dynarrestin, a Novel Inhibitor of Cytoplasmic DyneinCell Chemical Biology2018
Reinemann, Dana N. et al.Processive Kinesin-14 HSET Exhibits Directional Flexibility Depending on Motor TrafficCurrent Biology2018
Ricketts, Shea N. et al.Co-Entangled Actin-Microtubule Composites Exhibit Tunable Stiffness and Power-Law Stress RelaxationBiophysical Journal2018
Zhu, Yili et al.An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated ProteinBio-Protocol2018
Murray, John W. et al.Reduction of organelle motility by removal of potassium and other solutesPLoS ONE2017
Shim, Albert et al.Gliding Assay to Analyze Microtubule-based Motor Protein DynamicsBio-Protocol2017
Arellano-Santoyo, Hugo et al.A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase ActivityDevelopmental Cell2017
Reinemann, Dana N. et al.Collective Force Regulation in Anti-parallel Microtubule Gliding by Dimeric Kif15 Kinesin MotorsCurrent Biology2017
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 America2016
Shapira, Ofer et al.Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domainScientific Reports2016
Zitouni, Sihem et al.CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole BiogenesisCurrent Biology2016
Bartsch, Tobias F. et al.Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solutionNature Communications2016
Kim, Kyongwan et al.Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodesJournal of Applied Physics2015
Szyk, Agnieszka et al.Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferaseCell2014
Volkov, Vladimir A. et al.Preparation of segmented microtubules to study motions driven by the disassembling microtubule endsJournal of Visualized Experiments2014
Kitagawa, Mayumi et al.Cdk1 coordinates timely activation of MKlp2 kinesin with relocation of the chromosome passenger complex for cytokinesisCell Reports2014
Hawkins, Taviare L. et al.Mechanical properties of doubly stabilized microtubule filamentsBiophysical Journal2013
McVicker, Derrick P. et al.The nucleotide-binding state of microtubules modulates kinesin processivity and the ability of Tau to inhibit kinesin-mediated transportJournal of Biological Chemistry2011
Mori, Masashi et al.Intracellular Transport by an Anchored Homogeneously Contracting F-Actin MeshworkCurrent Biology2011
Mukhopadhyay, Aparna et al.Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesiclesJournal of Cell Science2011

Question 1:  Can TRITC rhodamine-labeled tubulin (Cat. # TL590M) be used to monitor tubulin dynamics in living cells?

Answer 1:  Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including TRITC rhodamine-tubulin, can be micro-injected into cells to study tubulin localization and dynamics in living cells.  Please see the brief protocol on the product datasheet (Cat. # TL590M) and these papers for guidance on micro-injecting cells with fluorescently-labeled proteins (Smilenov et al., 1999. Focal adhesion motility revealed in stationary fibroblasts. Science. 286, 1172-1174; Lopez-Lluch et al., 2001. Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils. Biochem. J. 357, 39-47; Lim and Danuser, 2009. Live cell imaging of F-actin dynamics via fluorescent speckle microscopy (FSM). J. Vis. Exp. 30, e1325, DOI: 10.3791/1325).

 

Question 2:  What is the best way to store TRITC rhodamine-labeled tubulin to maintain high activity?

Answer 2:  The recommended storage condition for the lyophilized tubulin product is 4°C in the dark 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.

 

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

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