Storage and Handling Instructions
Store at -80°C and protect from light
For research use only.
Shipping: shipped on dry ice
Storage Conditions: store at -80˚C immediately
Form: blue aqueous solution
Molecular Weight: ~110 kDa
Purity: >99% (SDS-PAGE)
Concentration: 20 mg/ml
Buffer Conditions: 50 mM K-Glutamate and 0.5 mM MgCl2 (pH 7.0)
Maximum Excitation/Emission: 649/670 nm
Labeling Stoichiometry: ~1.5 (check product label)
Shelf Life: check product label for expiration date
Tubulin is a labile molecule that loses activity within hours of being thawed. When handling Labeled Tubulin-Cy5, it is strongly recommended to follow the instructions detailed herein. Even perceived slight differences in handling can have a significant impact on product quality.
- store at -80°C and protect from light
- 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
- perform clarifying spin to remove protein aggregates
- visit our protocols page for common microtubule polymerization protocols
Immediately transfer Labeled Tubulin-Cy5 to -80°C upon receipt. Thaw only when ready to use by placing briefly in a 37°C water bath. Once the labeled tubulin is approximately halfway thawed, remove from the water bath and thaw to completion with gentle flicking. Immediately place thawed labeled tubulin on ice and continue to work on ice.
It is highly recommended to clarify the labeled tubulin after thawing to remove any protein aggregates. Centrifuge the labeled tubulin protein at 90k rpm for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100) and recover the supernatant on ice. The labeled tubulin is now ready for experimental use.
While repeated freeze/thaw cycles should be avoided, Labeled Tubulin-Cy5 can be aliquoted into smaller experimental batches with only minor loss of activity. Follow the instructions above to thaw and clarify the labeled tubulin protein. Working on ice, aliquot the labeled tubulin into experimental batches not less than 2 ul in volume. Flash freeze the experimental aliquots in liquid Nitrogen and store at -80˚C. Note that it is not recommended to dilute the labeled tubulin prior to refreezing. If larger handling volumes are desired, consider combining labeled tubulin with unlabeled tubulin prior to refreezing (depending on application).
Activity and Applications
Labeled Tubulin-Cy5 will polymerize into microtubules when supplemented with 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. Labeled tubulin is suitable for use in a variety of fluorescent experimental applications including live cell injection, and can be combined with unlabeled tubulin in generating fluorescent microtubules in vitro. Cycled Tubulin™ (Cat. No. 032005) is highly recommended as the unlabeled tubulin source in order to minimize tubulin heterogeneity, as labeled tubulin products are derived directly from Cycled Tubulin™. Visit our protocols page for more information.