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

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.


Cycled Tubulin™ is isolated by cycling bovine brain homogenate 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 2 years. 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.


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.


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 and loaded protein quantities are indicated.

Figure 2: Cycled Tubulin™ is polymerization-competent. Optical density (340 nm) of Cycled Tubulin™ that has undergone 1 (blue) or 2 (green) freeze/thaw cycles at 5 mg/ml in Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) supplemented with 1 mM GTP and 20% glycerol and incubated at 37°C. Distinct nucleation and polymerization phases are evident.

Cycled Tubulin turbidity assay

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

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

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