Tubulin protein (rhodamine): porcine brain

Tubulin protein (rhodamine): porcine brain
$0.00

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

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
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
Saper, Gadiel et al.Robotic end-to-end fusion of microtubules powered by kinesinScience Robotics2021ISSN 2470-9476
Lee, Gloria et al.Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamicsScience Advances2021ISSN 2375-2548
Hough, Cameron M. et al.Disassembly of microtubules by intense terahertz pulsesBiomedical Optics Express2021ISSN 2156--7085
Alfieri, Angus et al.Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruptionCurrent Biology2021ISSN 1879-0445
Habicht, Juri et al.UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin IIJournal of Cell Science2021ISSN 1477-9137
Kundu, Tanushree et al.Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth conesJournal of Cell Science2021ISSN 1477-9137
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020ISSN 1879-0445
Gaska, Ignas et al.The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule SlidingDevelopmental Cell2020ISSN 1878-1551
Saper, Gadiel et al.Kinesin-propelled label-free microtubules imaged with interference reflection microscopyNew Journal of Physics2020ISSN 1367-2630
Pyrpassopoulos, Serapion et al.Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule TrackBiophysical Journal2020
Kalra, Aarat P. et al.Investigation of the electrical properties of microtubule ensembles under cell-like conditionsNanomaterials2020ISSN 2079-4991
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020ISSN 1879-0445
Tuszynski, Jack A. et al.Microtubules as Sub-Cellular MemristorsScientific Reports2020ISSN 2045-2322
Chen, Keyu et al.Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubulesJournal of Cell Biology2020ISSN 1540-8140
Ricketts, Shea N. et al.Triggering Cation-Induced Contraction of Cytoskeleton Networks via MicrofluidicsFrontiers in Physics2020ISSN 2296-424X
Kaur, Simranpreet et al.Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A)Human Mutation2020ISSN 1098-1004
Adriaans, Ingrid E. et al.MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during AnaphaseCurrent Biology2020ISSN 1879-0445
Francis, Madison L. et al.Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton compositesSoft Matter2019ISSN 1744-6848
Faltova, Lenka et al.Crystal Structure of a Heterotetrameric Katanin p60:p80 ComplexStructure2019ISSN 1878-4186
Chudinova, Elena M. et al.On the interaction of ribosomal protein RPL22e with microtubulesCell Biology International2019ISSN 1095-8355
Fu, Meng meng et al.The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for MyelinationCell2019ISSN 1097-4172
Ricketts, Shea N. et al.Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule compositesScientific Reports2019ISSN 2045-2322
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
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
Leong, Su Ling et al.Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1Current Biology2019ISSN 0960-9822
Lopes, Joseph et al.Membrane mediated motor kinetics in microtubule gliding assaysScientific Reports2019ISSN 2045-2322
Fan, Yuanwei et al.The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus EndsCurrent Biology2018ISSN 0960-9822
Zhu, Yili et al.An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated ProteinBio-Protocol2018ISSN 2331--8325
Ricketts, Shea N. et al.Co-Entangled Actin-Microtubule Composites Exhibit Tunable Stiffness and Power-Law Stress RelaxationBiophysical Journal2018ISSN 1542-0086
Reinemann, Dana N. et al.Processive Kinesin-14 HSET Exhibits Directional Flexibility Depending on Motor TrafficCurrent Biology2018ISSN 0960-9822
Höing, Susanne et al.Dynarrestin, a Novel Inhibitor of Cytoplasmic DyneinCell Chemical Biology2018ISSN 2451-9448
Hilton, Nicholas A. et al.Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesisMolecular Microbiology2018ISSN 1365-2958
Aher, Amol et al.CLASP Suppresses Microtubule Catastrophes through a Single TOG DomainDevelopmental Cell2018ISSN 1878-1551
Ganguly, Anindya et al.Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4Developmental Cell2018ISSN 1878-1551
Colin, Alexandra et al.Actin-Network Architecture Regulates Microtubule DynamicsCurrent Biology2018ISSN 0960-9822
Romé, Pierre et al.A novel microtubule nucleation pathway for meiotic spindle assembly in oocytesJournal of Cell Biology2018ISSN 1540-8140
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
Rao, Lu et al.Combining structure–function and single-molecule studies on cytoplasmic dyneinMethods in Molecular Biology2018ISSN 1064-3745
Reinemann, Dana N. et al.Collective Force Regulation in Anti-parallel Microtubule Gliding by Dimeric Kif15 Kinesin MotorsCurrent Biology2017ISSN 0960-9822
Arellano-Santoyo, Hugo et al.A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase ActivityDevelopmental Cell2017ISSN 1878-1551
Shim, Albert et al.Gliding Assay to Analyze Microtubule-based Motor Protein DynamicsBio-Protocol2017ISSN 2331--8325
Murray, John W. et al.Reduction of organelle motility by removal of potassium and other solutesPLoS ONE2017ISSN 1932-6203
Zitouni, Sihem et al.CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole BiogenesisCurrent Biology2016ISSN 0960-9822
Shapira, Ofer et al.Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domainScientific Reports2016ISSN 2045-2322
Bartsch, Tobias F. et al.Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solutionNature Communications2016ISSN 2041-1723
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
Kim, Kyongwan et al.Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodesJournal of Applied Physics2015ISSN 1089-7550
Volkov, Vladimir A. et al.Preparation of segmented microtubules to study motions driven by the disassembling microtubule endsJournal of Visualized Experiments2014ISSN 1940-087X
Szyk, Agnieszka et al.Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferaseCell2014ISSN 1097-4172
Kitagawa, Mayumi et al.Cdk1 coordinates timely activation of MKlp2 kinesin with relocation of the chromosome passenger complex for cytokinesisCell Reports2014ISSN 2211-1247
Hawkins, Taviare L. et al.Mechanical properties of doubly stabilized microtubule filamentsBiophysical Journal2013ISSN 0006-3495
Mukhopadhyay, Aparna et al.Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesiclesJournal of Cell Science2011ISSN 0021-9533
Mori, Masashi et al.Intracellular Transport by an Anchored Homogeneously Contracting F-Actin MeshworkCurrent Biology2011
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 Chemistry2011ISSN 0021-9258

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.