AMCA labeled microtubules formed from AMCA labeled tubulin.
Product Uses Include
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 coefficient when 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.
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 TL440M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated AMCA 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: AMCA 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 AMCA tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of AMCA labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
V. VanDelinder and G.D. Bachand 2013. Photodamage and the importance of photoprotection in biomolecular-powered device applications. Anal. Chem. doi: 10.1021/ac403187g.
Question 1: Can AMCA-labeled tubulin (Cat. # TL440M) be used to monitor tubulin dynamics in living cells?
Answer 1: Yes, all of Cytoskeleton’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 (Cat. # TL440M 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 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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