X-rhodamine microtubules formed from X-rhodamine labeled tubulin.
Product Uses Include
Bovine brain tubulin (>99% pure, see Cat. # T240) 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.
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 TL620M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under green 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 (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 was run in a 4-20% SDS-PAGE system and photographed directly under green light illumination (B).
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.
Peña, A. et al. Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy. Structure 28, 450-457.e5 (2020).
Jiang, S. et al. Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7. Dev. Cell 49, 711-730.e8 (2019).
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Ho et al., 2011. Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis. Plant Cell. v 23, 2909–2923.
Question 1: Can X-rhodamine-labeled tubulin (Cat. # TL620M) be used to monitor tubulin dynamics in living cells?
Answer 1: Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including X-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. # TL620M 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-d 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 X-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%. 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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