Tubulin (Rhodamine labeled; porcine, micro-injctn)

On Arrival: 4°C

Question 1: Can TRITC rhodamine-labeled tubulin (Cat. #TL590M) be used to monitor tubulin dynamics in living cells?

Answer 1: Yes, all of Cytoskeleton&rsquo, 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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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 coefficientWhen 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 647^TM (Cat. #TL670M) labeled tubulins of the same quality.

Rhodamine tubulin protein purity determination. A 50 ug 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 ug of the same protein sample was run in a 4-20% SDS-PAGE system and photographed directly under UV illumination (B).