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DATASHEET: LABELED TUBULIN-BIOTIN-XX

Catalog Number: 033305
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 force-generating kinesin and dynein motor proteins. As such, immobilization of MTs onto solid surfaces such as glass coverslips and beads allows for the biophysical study of these molecular machines. Tubulin, the basic component of MTs, 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 labeled tubulin 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 Cycled Tubulin (Cat. No. 032005), thereby covalently linking the biomolecule 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 reactive biotin in its polymerized form and subjecting it to a final polymerization/depolymerization cycle. The final product demonstrates an affinity toward streptavidin-coated beads (Figure 1) and streptavidin-coated coverslips (Figure 2). Labeling stoichiometries are indicated on product labels. 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. The product is stable under these conditions for 1 year. Thaw only when ready to use by placing in a 37°C water bath followed by immediate placement on ice. 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.

Activity:

When supplemented with guanosine (GTP or GMPCPP) and warmed to 37°C, Labeled Tubulin-Biotin-XX will polymerize into MTs when above its critical concentration. It is recommended to combine Labeled Tubulin-Biotin-XX with unlabeled tubulin at a ratio of 1:50 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-Biotin-XX is supplied for use in a variety of experimental systems including:

  • optical traps and molecular tweezers
  • single molecule kinesin motor assays
  • tubulin affinity columns
  • in vitro nanoscale devices
Polymerization Protocol:

Dilute Labeled Tubulin-Biotin-XX to 0.04 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 2 mg/ml Cycled Tubulin (Cat. No. 032005), 1 mM DTT, and 1 mM guanosine (GTP or GMPCPP). If desired, Alexa Fluor®-labeled tubulin (Cat. Nos. 048805, 059405, and 064705) can be included at 0.2 mg/ml for visualization of the resulting microtubules. 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.

Technical Notes:
  • store at -80°C
  • 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-Biotin-XX binds to streptavidin-coated beads. Coomassie G250-stained protein gel of unbound and bound fractions of tubulin samples incubated with with Dynabeads® M-280 Streptavidin (Thermo Fisher 11205D). Cycled Tubulin serves as the non-biotinylated negative control. Labeled Tubulin-Biotin-XX 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 and 0.2 mg/ml Labeled Tubulin-Alexa Fluor® 647 +/- 0.04 mg/ml Labeled Tubulin-Biotin-XX. Microtubules polymerized with Labeled Tubulin-Biotin-XX are bound to the flow-cell surface.



References:

1. 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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