Tubulin protein (fluorescent HiLyte 488): porcine brain

Tubulin protein (fluorescent HiLyte 488): porcine brain

HiLyte Fluor™ 488 labeled microtubules formed from HiLyte Fluor™ 488 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 HiLyte Fluor™ 488 (HiLyte Fluor is a trademark of Anaspec Inc, CA) at random surface lysines. An activated ester of HiLyte Fluor™ 488 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 76,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. HiLyte Fluor™ 488 labeled tubulin can be detected using a filter set of 440-460 nm excitation and 500-520 emission. HiLyte Fluor™ 488 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), rhodamine (Cat. # TL590M), X-rhodamine (Cat. # TL620M) and HiLyte Fluor™ 647 (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 TL488M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated HiLyte Fluor™ 488 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: HiLyte Fluor™ 488 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 HiLyte Fluor™ 488 tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of AMCA 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
De Abreu, Isadora Rocha et al.A molecular analysis of substituted phenylethylamines as potential microtubule targeting agents through in silico methods and in vitro microtubule-polymerization activityScientific Reports 2023 13:12023ISSN 2045--2322
Singh, Rajendra K. et al.Coordinated Biophysical Stimulation of MSCs via Electromagnetized Au-Nanofiber Matrix Regulates Cytoskeletal-to-Nuclear Mechanoresponses and Lineage SpecificationAdvanced Functional Materials2023ISSN 1616--3028
Castrogiovanni, Cédric et al.Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubulesNature Communications 2022 13:12022ISSN 2041--1723
Masucci, Erin M. et al.Microtubule dynamics influence the retrograde biased motility of kinesin-4 motor teams in neuronal dendritesMolecular Biology of the Cell2022ISSN 1939-4586
Henty-Ridilla, Jessica L.Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) MicroscopyJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Sasanpour, Mehrzad et al.Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and MechanicsJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Baron, Desiree M et al.ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function.Cell reports2022ISSN 2211--1247
Cheng, Xianrui et al.Xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organizationJournal of Visualized Experiments2021ISSN 1940-087X
Goldstein-Levitin, Alina et al.Intracellular functions and motile properties of bi-directional kinesin-5 cin8 are regulated by neck linker dockingeLife2021ISSN 2050-084X
Lee, Gloria et al.Active cytoskeletal composites display emergent tunable contractility and restructuringSoft Matter2021ISSN 1744-6848
Aher, Amol et al.CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin IncorporationCurrent Biology2020ISSN 1879-0445
Nolet, Felix E. et al.Nuclei determine the spatial origin of mitotic waveseLife2020ISSN 2050-084X
Chen, Keyu et al.Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubulesJournal of Cell Biology2020ISSN 1540-8140
Rodríguez-García, Ruddi et al.Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic MicrotubulesCurrent Biology2020ISSN 1879-0445
Farhadi, Leila et al.Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal networkSoft Matter2020ISSN 1744-6848
Nakos, Konstantinos et al.Regulation of microtubule plus end dynamics by septin 9Cytoskeleton2019ISSN 1949-3592
Guedes-Dias, Pedro et al.Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the PresynapseCurrent Biology2019ISSN 0960-9822
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
Fan, Yuanwei et al.The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus EndsCurrent Biology2018ISSN 0960-9822
Colin, Alexandra et al.Actin-Network Architecture Regulates Microtubule DynamicsCurrent 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
Melo, Esther et al.HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease ModelEBioMedicine2018ISSN 2352-3964
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
Zhang, Rui et al.Interplay of structure, elasticity, and dynamics in actin-based nematic materialsProceedings of the National Academy of Sciences of the United States of America2017ISSN 1091-6490
Kandel, Mikhail E. et al.Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference MicroscopyACS Nano2017ISSN 1936-086X
Arellano-Santoyo, Hugo et al.A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase ActivityDevelopmental Cell2017ISSN 1878-1551
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
Taberner, Núria et al.Reconstituting Functional Microtubule-Barrier InteractionsMethods in Cell Biology2014ISSN 0091-679X
DeBerg, Hannah A. et al.Motor domain phosphorylation modulates kinesin-1 transportJournal of Biological Chemistry2013ISSN 0021-9258
Rostovtseva, Tatiana K. et al.Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel *Journal of Biological Chemistry2012ISSN 0021--9258
Hara, Masatoshi et al.Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factorNature Communications2012ISSN 2041-1723


Question 1:  Can HiLyte Fluor™ 488-labeled tubulin (Cat. # TL488M) be used to monitor tubulin dynamics in living cells?

Answer 1:  Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including HiLyte Fluor™ 488-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. # TL488M 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-d 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 HiLyte Fluor™ 488-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%.  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