Datasheet
Biotin-XX-Tubulin
| Catalog Number | Volume | Quantity |
|---|---|---|
| 033305 | 5 ul | 0.1 mg |
Made in the USA
Store at -80°C
For research use only.
Purity: >99%
Source: bovine
Molecular Weight: ~110 kDa
Labeling Stoichiometry: ~4.0 (check product label)
Form: clear aqueous solution
Concentration: 20 mg/ml
Buffer Conditions: 50 mM K-Glutamate and 0.5 mM MgCl2 (pH 7.0)
Shipping: shipped on dry ice
Storage Conditions: store at -80˚C immediately
Shelf Life: check product label for expiration date
Background
The microtubule network is a dynamic, force-generating cytoskeletal system essential for a number of basic cellular processes. Microtubules also serve as a track for force-generating kinesin and dynein motor proteins. As such, immobilization of microtubules onto solid surfaces such as glass coverslips and beads allows for the biophysical study of these molecular machines. Tubulin protein, the basic component of microtubules, can be functionalized by covalent linkage with Biotin-XX, a long-chain biotin derivative. Such modification must be performed in a way that maintains tubulin polymerization competency and functionality. The resulting biotinylated tubulin protein is useful in a number of applications ranging from optical traps to tubulin affinity columns.
Material
Labeled Tubulin-Biotin-XX is generated by reacting Biotin-XX succinimidyl ester with >99% pure tubulin protein, thereby covalently linking the biomolecule to random tubulin surface lysines. Polymerization competency is maintained during the labeling process by reacting the biotin with polymerized microtubules and subjecting the tubulin protein to a final polymerization/depolymerization cycle. The final product demonstrates an affinity toward streptavidin-coated beads (Figure 1) and streptavidin-coated coverslips (Figure 2). Specific labeling stoichiometries are indicated on the product label. The product is cryopreserved at 20 mg/ml in 50 mM K-Glutamate and 0.5 mM MgCl2 (pH=7.0).
Storage and Handling
Immediately transfer Labeled Tubulin-Biotin-XX 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 biotinylated tubulin after thawing to remove any protein aggregates by centrifugation at 90k rpm (350k x g) for 5 minutes at 4°C. If desired, Labeled Tubulin-Biotin-XX can be aliquoted into smaller experimental batches, frozen in liquid Nitrogen, and stored at -80°C with minor loss of polymerization competency. Avoid repeated freeze-thaw cycles. View detailed storage and handling instructions.
Activity and Applications
Labeled Tubulin-Biotin-XX 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. Biotinylated tubulin is suitable for use in a variety of experimental applications and can be combined with other labeled or unlabeled tubulin proteins (Cycled Tubulin™ highly recommended; Cat. No. 032005) 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.
- optical traps and molecular tweezers
- single molecule kinesin and dynein motor assays
- tubulin affinity columns
- in vitro nanoscale devices
Figure 1:
Labeled Tubulin-Biotin-XX binds to streptavidin-coated beads. Coomassie G250-stained protein gel of unbound and bound fractions of tubulin protein samples incubated with Dynabeads® M-280 Streptavidin (Thermo Fisher 11205D). Cycled Tubulin™ (Cat. No. 032005) serves as the non-biotinylated negative control. Labeled Tubulin-Biotin-XX (Cat. No. 033305) fractionates with the streptavidin-coated beads.
Figure 2:
Labeled Tubulin-Biotin-XX binds to streptavidin-coated coverslips.
Fluorescent images of microtubules incubated in streptavidin-coated flow cells. Microtubules were polymerized with 1.8 mg/ml Cycled Tubulin™ (Cat. No. 032005) and 0.2 mg/ml Labeled Tubulin-Alexa Fluor® 647 (Cat. No. 064705) +/- 0.05 mg/ml Labeled Tubulin-Biotin-XX (Cat. No. 033305). Microtubules polymerized with Labeled Tubulin-Biotin-XX are bound to the flow-cell surface.
References
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- Malcos, J.L. and Hancock, W.O. Engineering tubulin: microtubule functionalization approaches for nanoscale device applications. Appl. Microbiol. Biotechnol. 90(1), 1-10 (2011).