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Conjugation of fluorophores to tubulin

Nature Methods volume 2, pages 299–303 (2005)Cite this article

The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton1,2,3,4 and has become a powerful approach for studying the distribution and dynamics of these components5,6,7. Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues8,9,10,11. Fluorophores containing amine or thiol reactive groups are used to selectively modify lysine or cysteine residues, respectively. It is essential that derivatization of the protein not result in loss of function. Methods for selecting active protein and removing unconjugated fluorophore are usually used after derivatization. The choice of fluorophore at any given wavelength of fluorescence emission should optimize fluorescence intensity and photostability. Tubulin is labeled in polymeric form to protect residues that are important for assembly and can be successfully conjugated with fluorescent derivatives of amine-reactive succinimidyl esters. Functional tubulin is selected by cycles of polymerization and depolymerization. By modifying the ratio of reactants, the Cy3-labeling protocol can also be used to derivatize tubulin with Cy3.5, Cy5, tetramethylrhodamine, X-rhodamine, Oregon Green 488, and Alexa 488, 568 and 594 succinimidyl esters. Although fluorescent fusion proteins have become widely used in imaging studies, fluorophore-labeled proteins remain the most versatile and direct tools for studying the dynamics and function of the cytoskeleton. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.

  • Cy3 bis-reactive dye pack: 0.2 mg per vial (Amersham Pharmacia Biotech)

  • DMSO, anhydrous (Sigma)

  • Twice-cycled microtubule protein (MTP) preparation, isolated as described in ref. 9 (stored as pellets frozen at −80 °C)

  • 100 mM MgCl2

  • 100 mM sodium GTP

  • 1 M sodium PIPES (pH 6.9)

  • PM buffer: 0.1 M PIPES (pH 6.9), 1 mM MgCl2

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Figure 1: Speckle analysis reveals microtubule treadmilling in a CHO-K1 cytoplast lacking the centrosome.

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Authors and Affiliations

  1. Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, 60611, Illinois, USA

    John Peloquin, Yulia Komarova & Gary Borisy

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  1. John Peloquin
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  2. Yulia Komarova
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Correspondence to Gary Borisy.

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Peloquin, J., Komarova, Y. & Borisy, G. Conjugation of fluorophores to tubulin. Nat Methods 2, 299–303 (2005). https://doi.org/10.1038/nmeth0405-299

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