Tubulin protein (rhodamine): porcine brain

Tubulin protein (rhodamine): porcine brain

Rhodamine labeled microtubules formed from rhodamine labeled tubulin.


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

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.

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).


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

Lee, G. et. Al, Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics DOI: 10.1126/sciadv.abe4334 (2021)

Pyrpassopoulos, Serapion et al. “Modulation of Kinesin's Load-Bearing Capacity by Force Geometry and the Microtubule Track.” Biophysical journal vol. 118,1 (2020): 243-253. doi:10.1016/j.bpj.2019.10.045

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

Francis, Madison L et al. “Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites.” Soft matter vol. 15,44 (2019): 9056-9065. doi:10.1039/c9sm01550g

Grueb, S.S et al. "The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain." Sci Rep 9, 5352 (2019). https://doi.org/10.1038/s41598-019-41731-y

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

Zhu, Yili et al. “An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein.” Bio-protocol vol. 8,23 (2018): e3110. doi:10.21769/BioProtoc.3110

Hawkins et al., 2012. Perturbations in Microtubule Mechanics from Tubulin Preparation. Cell. Mol. Bioengineer. v 5, pp 227-238.

Nakajima et al., 2012. Enhancement of tubulin polymerization by Cl−-induced blockade of intrinsic GTPase. Biochem. Biophys. Res. Commun. doi:http://dx.doi.org/10.1016/j.bbrc.2012.07.072.

Mukhopadhyay et al., 2011. Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles. J. Cell Sci. v 124, pp 765-775.

Mori et al., 2011. Intracellular Transport by an Anchored Homogeneously Contracting F-Actin Meshwork. Curr. Biol. v 21, pp 606-611.

McVicker et al., 2011. The Nucleotide-binding State of Microtubules Modulates Kinesin Processivity and the Ability of Tau to Inhibit Kinesin-mediated Transport. J. Biol. Chem. v 286, pp 42873-42880.

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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
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
Lee, Gloria et al.Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamicsScience Advances2021ISSN 2375-2548
Saper, Gadiel et al.Robotic end-to-end fusion of microtubules powered by kinesinScience Robotics2021ISSN 2470-9476
Kundu, Tanushree et al.Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth conesJournal of Cell Science2021ISSN 1477-9137
Hough, Cameron M. et al.Disassembly of microtubules by intense terahertz pulsesBiomedical Optics Express2021ISSN 2156--7085
Adriaans, Ingrid E. et al.MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during AnaphaseCurrent Biology2020ISSN 1879-0445
Kalra, Aarat P. et al.Investigation of the electrical properties of microtubule ensembles under cell-like conditionsNanomaterials2020ISSN 2079-4991
Saper, Gadiel et al.Kinesin-propelled label-free microtubules imaged with interference reflection microscopyNew Journal of Physics2020ISSN 1367-2630
Gaska, Ignas et al.The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule SlidingDevelopmental Cell2020ISSN 1878-1551
Kaur, Simranpreet et al.Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A)Human Mutation2020ISSN 1098-1004
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020ISSN 1879-0445
Tuszynski, Jack A. et al.Microtubules as Sub-Cellular MemristorsScientific Reports2020ISSN 2045-2322
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020ISSN 1879-0445
Ricketts, Shea N. et al.Triggering Cation-Induced Contraction of Cytoskeleton Networks via MicrofluidicsFrontiers in Physics2020ISSN 2296-424X
Chen, Keyu et al.Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubulesJournal of Cell Biology2020ISSN 1540-8140
Fu, Meng meng et al.The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for MyelinationCell2019ISSN 1097-4172
Chudinova, Elena M. et al.On the interaction of ribosomal protein RPL22e with microtubulesCell Biology International2019ISSN 1095-8355
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
Faltova, Lenka et al.Crystal Structure of a Heterotetrameric Katanin p60:p80 ComplexStructure2019ISSN 1878-4186
Leong, Su Ling et al.Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1Current Biology2019ISSN 0960-9822
Ricketts, Shea N. et al.Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule compositesScientific Reports2019ISSN 2045-2322
Francis, Madison L. et al.Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton compositesSoft Matter2019ISSN 1744-6848
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
Lopes, Joseph et al.Membrane mediated motor kinetics in microtubule gliding assaysScientific Reports2019ISSN 2045-2322
Romé, Pierre et al.A novel microtubule nucleation pathway for meiotic spindle assembly in oocytesJournal of Cell Biology2018ISSN 1540-8140
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
Rao, Lu et al.Combining structure–function and single-molecule studies on cytoplasmic dyneinMethods in Molecular Biology2018ISSN 1064-3745
Höing, Susanne et al.Dynarrestin, a Novel Inhibitor of Cytoplasmic DyneinCell Chemical Biology2018ISSN 2451-9448
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Ganguly, Anindya et al.Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4Developmental Cell2018ISSN 1878-1551
Hilton, Nicholas A. et al.Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesisMolecular Microbiology2018ISSN 1365-2958
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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.