Datasheet

LABELED TUBULIN-CY5

Made in the USA

Catalog Number Volume Quantity
065005 - 0.1 mg 5 ul 0.1 mg
065005 - 0.5 mg 25 ul 0.5 mg
065005 - 1.0 mg 50 ul 1.0 mg

Store at -80°C and protect from light

For research use only.

Purity: >99%
Source: bovine
Molecular Weight: ~110 kDa
Maximum Excitation/Emission: 649/670 nm
Labeling Stoichiometry: ~1.5 (check product label)
Form: blue 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 kinesin and dynein motor proteins. As such, visualization of microtubules in real time, both in cells and in vitro, is critical in understanding cellular function and human disease. Tubulin protein, the basic component of microtubules, can be functionalized for visualization by covalent linkage with a fluorescent dye. Such modification must be performed in a way that maintains tubulin polymerization competency and functionality. The resulting labeled tubulin protein is useful in a number of applications ranging from live cell injection/imaging to in vitro nanoscale devices.

Material

Labeled Tubulin-Cy5 is generated by reacting Cy5 mono-reactive NHS ester with >99% pure tubulin protein, thereby covalently linking the dye to random tubulin surface lysines. Polymerization competency is maintained during the labeling process by reacting the tubulin protein with the dye in its polymerized form and subjecting it to a final polymerization/depolymerization cycle. The final product displays maximum absorption at 280 nm and 649 nm and a labeling stoichiometry ([dye]/[tubulin]) of ~1.5 as determined by spectroscopic analysis (Figure 1). Specific labeling stoichiometries are indicated on the product label. Labeled Tubulin-Cy5 is commonly visualized with a Cy5 filter set with maximum excitation/emission wavelengths of 649/670 nm (Figure 2). 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-Cy5 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 labeled 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, Labeled Tubulin-Cy5 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 and protect from light. View detailed storage and handling instructions.

Activity and Applications

Labeled Tubulin-Cy5 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. Labeled tubulin protein is suitable for use in a variety of fluorescent experimental applications including live cell injection, and can be combined with unlabeled tubulin protein 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 reagents are derived directly from Cycled Tubulin™. 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.

  • in vitro microtubule gliding assays
  • single molecule kinesin and dynein motor assays
  • speckle microscopy
  • live cell injection
  • in vitro nanoscale devices
  • TIRF, STORM, SIM, STED, TPE, confocal, and widefield microscopy
Cy5 - Absorbance Graph

Figure 1:

Labeled Tubulin-Cy5 displays maximum absorption at 280 nm and 649 nm. Spectroscopic analysis reveals a peak at 280 nm and 649 nm, indicating absorption by the tubulin protein and Cy5 dye, respectively.

Figure 2:

Labeled Tubulin-Cy5 is commonly visualized with a Cy5 filter set.
Taxol-stabilized microtubules at 5 mg/ml total tubulin protein with Labeled Tubulin-Cy5 (Cat. No. 065005) and Cycled Tubulin™ (Cat. No. 032005) at a 1:5 ratio.  Image acquired on a widefield fluorescence microscope.

Taxol stabilized microtubules polymerized from Cy5 labeled tubulin protein

References

  1. Keith, C.H., Feramiscoa, J.R., Shelanski, M. Direct visualization of fluorescein labeled microtubules in vitro and in microinjected fibroblasts. J. Cell Biol. 88, 234–240 (1981).
  2. Panchuk-Voloshina, N., Haugland, R.P., Bishop-Stewart, J., Bhalgat, M.K., Millard, P.J., Mao, F., Leung, W.Y. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188 (1999).
  3. Malcos, J.L. and Hancock, W.O. Engineering tubulin: microtubule functionalization approaches for nanoscale device applications. Appl. Microbiol. Biotechnol. 90(1), 1-10 (2011).