Cat. # TL334M

Product Uses Include

• Laser based applications
• Monitoring microtubule dynamcs in living cells by microinjection
• 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 rhodamine at random surface lysines. An activated ester of X-rhodamine (ROX) 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 70,000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. X-rhodamine labeled tubulin can be detected using a filter set of 560-580 nm excitation and 605-620 emission. X-rhodamine tubulin is in a versatile, stable and easily shipped format. When dissolved in G-PEM, it is ready for micro-injection, when dissolved in G-PEM with 5% glycerol, it is ready for in vitro polymerization. Cytoskeleton, Inc. also offers rhodamine and fluorescein labeled tubulin of the same quality (for example Cat.# TL331M and T332M).

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 TL334M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated 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 (Figure 1 B).

Figure 1: X-rhodamine 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 after rhodamine conjugation was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).

Examples of publications where this product was used

X-rhodamine is a new product as of June 2006, there are no citations relating to this product at this time.