DATASHEET: CYCLED TUBULIN™ (>99%)
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
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.
- 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 2: Cycled Tubulin™ is polymerization-competent. Absorbance (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.
Comparison with Lyophilized Tubulin (Cat. No. 142001):
|Specifications||Cycled Tubulin™||Lyophilized Tubulin|
|Pricing||< $150/mg||< $105/mg|
|Shipping Method||FedEx Overnight on Dry Ice||FedEx 2 Day Envelope|
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