HiLyte Fluor™ 647labeled microtubules formed from HiLyte Fluor™ 647 labeled tubulin.
Product Uses Include
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked HiLyte Fluor™ 647 (HiLyte Fluor is a trademark of Anaspec Inc, CA) at random surface lysines. An activated ester of HiLyte Fluor™ 647 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 250,000M-1cm-1). Final labeling stoichiometry is 0.2 to 0.7 dyes per tubulin heterodimer. HiLyte Fluor™ 647 labeled tubulin can be detected using a filter set of 600-630 nm excitation and 660-680 emission. HiLyte Fluor™ 647 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), rhodamine (Cat. # TL590M), X-rhodamine (Cat. # TL620M) labeled tubulins.
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 TL670M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated HiLyte Fluor™ 670 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: HiLyte Fluor™ 647 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 525-625nm illumination (B).
The biological activity of HiLyte Fluor™ 647 tubulin is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of HiLyte Fluor™ 647 labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
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Farhadi, Leila et al. “Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network.” Soft matter vol. 16,31 (2020): 7191-7201. doi:10.1039/c9sm02400j
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).
Li, F, Pan, J, Choi, JH. Local direction change of surface gliding microtubules. Biotechnology and Bioengineering. 2019; 116: 1128– 1138. https://doi.org/10.1002/bit.26933
Nakos, Konstantinos et al. “Regulation of microtubule plus end dynamics by septin 9.” Cytoskeleton (Hoboken, N.J.) vol. 76,1 (2019): 83-91. doi:10.1002/cm.21488
Chen, Y., Su, Q. P., Sun, Y., & Yu, L. (2018). Visualizing autophagic lysosome reformation in cells using in vitro reconstitution systems. Current Protocols in Cell Biology, 78, 11.24.1– 11.24.15. doi: 10.1002/cpcb.44
Dumont, E., Do, C. & Hess, H. Molecular wear of microtubules propelled by surface-adhered kinesins. Nature Nanotech 10, 166–169 (2015). https://doi.org/10.1038/nnano.2014.334
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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. 23, 2909–2923.
Question 1: Can HiLyte Fluor™ 647-labeled tubulin (Cat. # TL670M) 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. # TL670M 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 HiLyte Fluor™ 647-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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