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DATASHEET: LABELED TUBULIN-ALEXA FLUOR® 647

Catalog Number: 064705
Source: Bovine Brain
Store at -80°C
Background:

The microtubule (MT) network is a dynamic, force-generating cytoskeletal system essential for a number of basic cellular processes. MTs also serve as a track for kinesin and dynein motor proteins. As such, visualization of MTs in real time, both in cells and in vitro, is critical in understanding cellular function and human disease. Tubulin, the basic component of MTs, 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 is useful in a number of applications ranging from live cell injection/imaging to in vitro nanoscale devices.

Material:

Labeled Tubulin-Alexa Fluor® 647 is generated by reacting Alexa Fluor® 647 succinimidyl ester (Thermo Fisher A20106) with Cycled Tubulin™ (Cat. No. 032005), thereby covalently linking the dye to random tubulin surface lysines. Cycled Tubulin™ is >99% pure and polymerization competent. These properties are maintained during the labeling process by reacting Cycled Tubulin™ 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 651 nm and a labeling stoichiometry ([dye]/[tubulin]) of 0.5-2.0 as determined by spectroscopic analysis (Figure 1). Specific labeling stoichiometries are indicated on the product label. Labeled Tubulin-Alexa Fluor® 647 is commonly visualized with a Cy5 filter set with maximum excitation/emission wavelengths of 651/672 nm. 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-Alexa Fluor® 647 to -80°C upon receipt. The product is stable under these conditions for 2 years. Thaw only when ready to use by placing in a 37°C water bath followed by immediate placement on ice. If desired, Labeled Tubulin-Alexa Fluor® 647 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.

Activity:

When supplemented with guanosine (GTP or GMPCPP) and warmed to 37°C, Labeled Tubulin-Alexa Fluor® 647 will polymerize into MTs when above its critical concentration. It is recommended to combine Labeled Tubulin-Alexa Fluor® 647 with unlabeled tubulin at a ratio of 1:9 to a total tubulin concentration of 2 mg/ml. 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.

Uses:

Labeled Tubulin-Alexa Fluor® 647 is supplied for use in fluorescent experimental systems including:

  • TIRF, STORM, SIM, STED, TPE, confocal, and widefield microscopy applications
  • in vitro MT gliding assays
  • single molecule kinesin motor assays
  • speckle microscopy
  • in vitro nanoscale devices
  • injection into live cells
Polymerization Protocol:

Dilute Labeled Tubulin-Alexa Fluor® 647 to 0.2 mg/ml with Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) and supplement with 1.8 mg/ml Cycled Tubulin™ (Cat. No. 032005), 1 mM DTT, and 1 mM guanosine (GTP or GMPCPP). Incubate on ice for 5 minutes, then transfer to a 37°C water bath for 1 hour. If polymerized with GMPCPP or protected with Taxol, the resulting MTs will be stable at room temperature for several days. Do not place polymerized MTs on ice. Protect polymerized MTs from light.

Technical Notes:
  • store at -80°C
  • 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
  • regard tubulin concentration, temperature, and guanosine addition when polymerizing
  • do not place polymerized MTs on ice

Figure 1: Labeled Tubulin-Alexa Fluor® 647 displays maximum absorption at 280 nm and 651 nm. Spectroscopic analysis reveals a peak at 280 nm and 651 nm, indicating absorption by tubulin and Alexa Fluor® 647, respectively.


alexa-647-tubulin

Comparison with other labeled tubulins:

Specifications Alexa Fluor® 488 Alexa Fluor® 594 Alexa Fluor® 647
Cat. No. 048805 059405 064705
Purity >99% >99% >99%
Cycled Yes Yes Yes
Ex/Em 494/517 nm 590/617 nm 651/672 nm
Storage Method Cryopreserved Cryopreserved Cryopreserved
Shipping Method FedEx Overnight on Dry Ice FedEx Overnight on Dry Ice FedEx Overnight on Dry Ice
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).


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