For research use only.
Molecular Weight: ~110 kDa
Form: clear aqueous solution
Concentration: 20 mg/ml
Buffer Conditions: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2 (pH 6.8)
Shipping: shipped on dry ice
Storage Conditions: store at -80˚C immediately
Shelf Life: check product label for expiration date
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. Microtubules 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, microtubules have inherent polarity with distinct “+” (β-tubulin exposed) and “-” (α-tubulin exposed) ends. Another critical feature of microtubules is their dynamic instability, a consequence of the GTPase activity of tubulin. This property confers force-generating capabilities to microtubules 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 tubulin protein is >99% pure (Figure 1) and polymerization competent (Figure 2). Cycled Tubulin™ is cryopreserved at 20 mg/ml in Tubulin PEM Buffer (also known as BRB80; 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. Thaw only when ready to use by placing briefly in a 37°C water bath followed by immediate placement on ice. Clarify the tubulin after thawing to remove any protein aggregates by centrifugation at 90k rpm for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100). 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. View detailed storage and handling instructions.
Activity and Applications
Cycled Tubulin™ will polymerize into microtubules when supplemented with guanosine-5’-triphosphate (GTP), warmed to 37˚C, and kept above its critical concentration. Polymerization activity is detectable in a variety of experimental systems including fluorescence microscopy assays, turbidity assays, and GTPase assays. Cycled Tubulin™ is suitable for use in a variety of cell-free experimental applications and can be combined with fluorescent or biotinylated tubulin proteins in generating microtubules in vitro. Visit our protocols page for common microtubule polymerization protocols, including the generation of short, rigid microtubules stabilized by GMPCPP or long, flexible microtubules stabilized by taxol.
- structural analysis by X-ray crystallography and electron microscopy
- drug discovery by high-throughput screening
- in vitro biochemical and biophysical approaches
Cycled Tubulin™ is >99% pure. Coomassie G250-stained protein gel of Cycled Tubulin™ separated by SDS-PAGE. The tubulin protein appears as a single species migrating at ~55 kDa. Molecular weight markers and loaded protein quantities are indicated.
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. 032003; 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.
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