Rhodamine labeled microtubules formed from rhodamine labeled tubulin.
Product Uses Include
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked rhodamine at random surface lysines. An activated ester of 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 64,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. rhodamine labeled tubulin can be detected using a filter set of 530-550 nm excitation and 580-600 emission. 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™ 488 (Cat. # TL488M), X-rhodamine (Cat. # TL620M) and HiLyte Fluor™ 647TM (Cat. # TL670M) labeled tubulins of the same quality.
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 TL590M 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: 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 was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).
The biological activity of rhodamine tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of rhodamine labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
Hawkins et al., 2012. Perturbations in Microtubule Mechanics from Tubulin Preparation. Cell. Mol. Bioengineer. v 5, pp 227-238.
Nakajima et al., 2012. Enhancement of tubulin polymerization by Cl−-induced blockade of intrinsic GTPase. Biochem. Biophys. Res. Commun. doi:http://dx.doi.org/10.1016/j.bbrc.2012.07.072.
Mukhopadhyay et al., 2011. Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles. J. Cell Sci. v 124, pp 765-775.
Mori et al., 2011. Intracellular Transport by an Anchored Homogeneously Contracting F-Actin Meshwork. Curr. Biol. v 21, pp 606-611.
McVicker et al., 2011. The Nucleotide-binding State of Microtubules Modulates Kinesin Processivity and the Ability of Tau to Inhibit Kinesin-mediated Transport. J. Biol. Chem. v 286, pp 42873-42880.
Question 1: Can TRITC rhodamine-labeled tubulin (Cat. # TL590M) be used to monitor tubulin dynamics in living cells?
Answer 1: Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including TRITC rhodamine-tubulin, can be micro-injected into cells to study tubulin localization and dynamics in living cells. Please see the brief protocol on the product datasheet (Cat. # TL590M) and these papers for guidance on micro-injecting cells with fluorescently-labeled proteins (Smilenov et al., 1999. Focal adhesion motility revealed in stationary fibroblasts. Science. 286, 1172-1174; Lopez-Lluch et al., 2001. Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils. Biochem. J. 357, 39-47; Lim and Danuser, 2009. Live cell imaging of F-actin dynamics via fluorescent speckle microscopy (FSM). J. Vis. Exp. 30, e1325, DOI: 10.3791/1325).
Question 2: What is the best way to store TRITC rhodamine-labeled tubulin to maintain high activity?
Answer 2: The recommended storage condition for the lyophilized tubulin product is 4°C in the dark with desiccant to maintain humidity at <10% humidity. Under these conditions the protein is stable for 6 months. Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months. However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture. Therefore we recommend storing at 4°C. If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. After reconstituting the protein as directed, the concentrated protein in G-PEM buffer should be aliquoted, snap frozen in liquid nitrogen and stored at -70°C (stable for 6 months). NOTE: It is very important to snap freeze the tubulin in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
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