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
Bovine brain tubulin (>99% pure, see Cat. # TL238) has been modified to contain covalently linked X-rhodamine at random surface lysines. An activated ester of X-rhodamine 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 66,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. X-rhodamine labeled tubulin can be detected using a filter set of 540-560 nm excitation and 610-630 emission. X-rhodamine 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™ 488TM (Cat. # TL488M), rhodamine (Cat. # TL590M) and HiLyte Fluor™ 647TM (Cat. # TL670M) labeled tubulins of the same quality.
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 TL440M is >99% pure tubulin. Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated X-rhodamine 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.
Biological Activity
The biological activity of X-rhodamine tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of X-rhodamine labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
