Tubulin protein (X-rhodamine): porcine brain

Tubulin protein (X-rhodamine): porcine brain


X-rhodamine microtubules formed from X-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 X-rhodamine at random surface lysines. An activated ester of X-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 66,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. X-rhodamine labeled tubulin can be detected using a filter set of 540-560 nm excitation and 610-630 emission. X-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™ 488TM (Cat. # TL488M), rhodamine (Cat. # TL590M) 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 TL620M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under green light. Any unincorporated X-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: X-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 green light illumination (B).

Biological Activity
The biological activity of X-rhodamine tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of X-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
Liu, Tianyang et al.Mechanochemical tuning of a kinesin motor essential for malaria parasite transmissionNature Communications 2022 13:12022ISSN 2041--1723
Gibson, James M et al.Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitmenteLife2022ISSN 2050-084X
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
Qiu, Rongde et al.Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domainCurrent Biology2021ISSN 1879-0445
Zhernov, Ilia et al.Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional DiversityCurrent Biology2020ISSN 1879-0445
McElmurry, Kristi et al.Dynein-mediated microtubule translocation powering neurite outgrowth in chick and Aplysia neurons requires microtubule assemblyJournal of Cell Science2020ISSN 1477-9137
Peña, Alejandro et al.Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron MicroscopyStructure2020ISSN 1878-4186
Nakos, Konstantinos et al.Regulation of microtubule plus end dynamics by septin 9Cytoskeleton2019ISSN 1949-3592
Jiang, Shuo et al.Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7Developmental Cell2019ISSN 1878-1551
Sladewski, Thomas E. et al.Recruitment of two dyneins to an mRNA-dependent bicaudal D transport complexeLife2018ISSN 2050-084X
Haase, Julian et al.Distinct Roles of the Chromosomal Passenger Complex in the Detection of and Response to Errors in Kinetochore-Microtubule AttachmentDevelopmental Cell2017ISSN 1878-1551
Maciejowski, John et al.Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome SegregationDevelopmental Cell2017ISSN 1878-1551
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


Question 1:  Can X-rhodamine-labeled tubulin (Cat. # TL620M) be used to monitor tubulin dynamics in living cells?

Answer 1:  Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including X-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. # TL620M 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 X-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%.  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