Product: Cycled Tubulin™
Catalog Number: 032005
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
Taxol Stabilized Microtubules (long and flexible)
Taxol-stabilized microtubules at 5 mg/ml total tubulin protein with Labeled Tubulin-Alexa Fluor® 647 (Cat. No. 064705) and Cycled Tubulin™ (Cat. No. 032005) at a 1:5 ratio. Images acquired on a widefield fluorescence microscope. View protocol.
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).
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.