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DATASHEET: CYCLED TUBULIN™ (>99%)

Catalog Number: 032005
Source: Bovine Brain
Store at -80°C
Background:

Tubulin, a highly conserved cytoskeletal protein, is required for several essential eukaryotic processes including intracellular transport, intercellular signaling, extracellular sensing, cell migration, and cell division. Tubulin (110 kDa) is a heterodimer of α- and β-tubulin (each 55 kDa), and polymerizes into higher order filaments termed microtubules (MTs). MTs measure 25 nm in diameter and have a persistence length of ~2 mm, incorporating ~1650 tubulin subunits per 1 μm. Given the asymmetry of tubulin dimers, MTs have inherent polarity with distinct “+” (β-tubulin exposed) and “-” (α-tubulin exposed) ends. Another critical feature of MTs is their dynamic instability, a consequence of the GTPase activity of tubulin. This property confers force-generating capabilities to MTs that are critical for cell division. For this reason, tubulin is a powerful target for the therapeutic intervention of neoplastic diseases such as cancers.

Material:

Cycled Tubulin™ is isolated by cycling bovine brain homogenates through conditions that promote tubulin polymerization/depolymerization in high salt buffers by an adaptation of the method of Castoldi and Popov (2003). The resulting product is >99% pure (Figure 1) and polymerization competent (Figure 2). Cycled Tubulin™ is cryopreserved at 20 mg/ml in Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8).

Storage and Handling:

Immediately transfer Cycled Tubulin™ to -80°C upon receipt. The product is stable under these conditions for 1 year. Thaw only when ready to use by placing in a 37°C water bath followed by immediate placement on ice. If desired, Cycled Tubulin™ can be aliquoted into smaller experimental batches, frozen in liquid Nitrogen, and stored at -80°C with minor loss of polymerization competency (Figure 2). Avoid repeated freeze-thaw cycles.

Activity:

When supplemented with guanosine (GTP or GMPCPP) and warmed to 37°C, Cycled Tubulin™ will polymerize into MTs when above its critical concentration. The recommended tubulin concentration for ensuring polymerization is 2 mg/ml.

Uses:

Cycled Tubulin™ is supplied for use in cell-free experimental systems including:

  • structural analysis by X-ray crystallography and electron microscopy
  • drug discovery by high-throughput screening
  • in vitro biochemical and biophysical approaches
Polymerization Protocol:

Dilute Cycled Tubulin™ to 2 mg/ml with Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) and supplement with 1 mM each DTT and guanosine (GTP or GMPCPP). Incubate on ice for 5 minutes, then transfer to a 37° C water bath for 1 hour. If polymerized with GMPCPP or protected with Taxol, the resulting MTs will be stable at room temperature for several days. Do not place polymerized MTs on ice.

Technical Notes:
  • store at -80°C
  • avoid repeated freeze-thaw cycles, refreeze in liquid Nitrogen if required
  • thaw only when ready to use at 37°C followed by immediate placement on ice
  • regard tubulin concentration, temperature, and guanosine addition when polymerizing
  • do not place polymerized MTs on ice

Figure 1: Cycled Tubulin™ is >99% pure. Coomassie G250-stained protein gel of Cycled Tubulin™ separated by SDS-PAGE. The tubulin appears as a single species migrating at ~55 kDa. Molecular weight markers (kDa) and loaded protein quantities are indicated.


Cycled Tubulin Purity PurSolutions

Figure 2: Cycled Tubulin™ is polymerization-competent. Optical Density (340 nm) of Cycled Tubulin™ at 5 mg/ml in Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) at 37°C. Distinct nucleation and polymerization phases are evident. GTP and DTT were included at 1 mM, and Glycerol was added to 5% v/v. Purple, product that has undergone one freeze-thaw cycle. Blue, product that has undergone two freeze-thaw cycles.

Cycled Tubulin Polymerization Turbidity Freeze-Thaw PurSolutions

Comparison with Lyophilized Tubulin (Cat. No. 142001):

Specifications Cycled Tubulin™ Lyophilized Tubulin
Cat No. 032005 142001
Purity >99% >99%
Cycled Yes No
Storage Method Cyropreserved Lyophilized
Storage Buffer PIPES Phosphate
Pricing < $150/mg < $105/mg
Shipping Methods FedEx Overnight on Dry Ice FedEx 2 Day Envelope
References:

1. Allen, C. & Borisy, G. G. Structural polarity and directional growth of microtubules of Chlamydomonas flagella. J. Mol. Biol. 90, 381–402 (1974).

2. Caplow, M., Ruhlen, R. L. & Shanks, J. The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice. J. Cell Biol. 127, 779–788 (1994).

3. Carlier, M. F., Hill, T. L. & Chen, Y. Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study. P Natl Acad Sci Usa 81, 771–775 (1984).

4. Castoldi, M. and Popov A. Purification of brain tubulin through two cycles of polymerization-depolymerization in high-molarity buffer. Protein Expression and Purification. 32, 83-88 (2003).

5. David-Pfeuty, T., Erickson, H. P. & Pantaloni, D. Guanosinetriphosphatase activity of tubulin associated with microtubule assembly. (1977).

6. Desai, A. & Mitchison, T. J. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117 (1997).

7. Evans, L., Mitchison, T. & Kirschner, M. Influence of the centrosome on the structure of nucleated microtubules. J. Cell Biol. 100, 1185–1191 (1985).

8. Mandelkow, E. M., Mandelkow, E. & Milligan, R. A. Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J. Cell Biol. 114, 977–991 (1991).

9. Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature (1984).

10. Nogales, E., Whittaker, M., Milligan, R. A. & Downing, K. H. High-Resolution Model of the Microtubule. Cell 96, 79–88 (1999).

11. Oosawa, F., 1922, Asakura, S.1927. Thermodynamics of the polymerization of protein. (1975).

12. Walker, R. A. et al. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J. Cell Biol. 107, 1437–1448 (1988).

13. Weisenberg, R. C., Deery, W. J. & Dickinson, P. J. Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules. Biochemistry 15, 4248–4254 (1976).


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