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HiLyte Fluor? 647labeled microtubules formed from HiLyte Fluor? 647 labeled tubulin.

Product Uses Include：

Laser based applications

Monitoring microtubule dynamcs in living cells

Speckle microscopy

Formation of fluorescent microtubules

Microscopy studies of MAP and microtubule associated motor activities

Nanotechnology

Material：

Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked HiLyte Fluor? 647 (HiLyte Fluor is a trademark of Anaspec Inc, CA) at random surface lysines. An activated ester of HiLyte Fluor? 647 was used to label the protein. Labeling stoichiometry was determined by spectroscopic measurement of protein and dye concentrations (dye extinction coefficient when protein bound is 250,000M-1cm-1). Final labeling stoichiometry is 0.2 to 0.7 dyes per tubulin heterodimer. HiLyte Fluor? 647 labeled tubulin can be detected using a filter set of 600-630 nm excitation and 660-680 emission. HiLyte Fluor? 647 tubulin is in a versatile, stable and easily shipped format. It is ready for micro-injection or in vitro polymerization. Cytoskeleton, Inc. also offers AMCA (Cat. # TL440M), HiLyte Fluor? 488 (Cat. # TL488M), rhodamine (Cat. # TL590M), X-rhodamine (Cat. # TL620M) labeled tubulins.

Purity：
      The protein purity of the tubulin used for labeling is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. The protein used for TL670M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated HiLyte Fluor? 670 dye would be visible in the dye front. No fluorescence is detected in the dye front, indicating that no free dye is present in the final product (Figure 1 B).

Figure 1: HiLyte Fluor? 647 tubulin protein purity determination. A 50 μg sample of unlabeled tubulin protein was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue (A). Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02). 20 μg of the same protein sample was run in a 4-20% SDS-PAGE system and photographed directly under 525-625nm illumination (B).

Biological Activity：
      The biological activity of HiLyte Fluor? 647 tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of HiLyte Fluor? 647 labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.