Tubulin (AMCA dye labeled; porcine, micro-injctn)

On Arrival: 4°C

Question 1: Can AMCA-labeled tubulin ((Cat. #TL440M) be used to monitor tubulin dynamics in living cells?

Answer 1: Yes, all of Cytoskeleton&rsquo, s fluorescently-labeled tubulins, including AMCA-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 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-dC2-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 AMCA-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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Porcine brain tubulin (>99% pure, see Cat. #T240) has been modified to contain covalently linked 7-amino-4-methyl coumarin-3-acetic acid (AMCA) at random surface lysines. An activated ester of X-AMCA was used to label the protein. Labeling stoichiometry was determined by spectroscopic measurement of protein and dye concentrations (dye extinction coefficientWhen protein bound is 40, 000M^-1cm^-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. AMCA labeled tubulin can be detected using a filter set of 350-370 nm excitation and 430-450 emission. AMCA tubulin is in a versatile, stable and easily shipped format. It is ready for micro-injection or in vitro polymerization. Cytoskeleton, Inc. also offers HiLyte Fluor 488 (Cat. #TL488M), rhodamine (Cat. #TL590M), X-rhodamine (Cat. #TL620M) and HiLyte Fluor 647 (Cat. #TL670M) labeled tubulins of the same quality.

Figure 1. AMCA 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).