HiLyte Fluor™ 488 labeled microtubules formed from HiLyte Fluor™ 488 labeled tubulin.
Product Uses Include
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified to contain covalently linked HiLyte Fluor™ 488 (HiLyte Fluor is a trademark of Anaspec Inc, CA) at random surface lysines. An activated ester of HiLyte Fluor™ 488 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 76,000M-1cm-1). Final labeling stoichiometry is 1-2 dyes per tubulin heterodimer. HiLyte Fluor™ 488 labeled tubulin can be detected using a filter set of 440-460 nm excitation and 500-520 emission. HiLyte Fluor™ 488 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), 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 TL488M is >99% pure tubulin (Figure 1 A). Labeled protein is run on an SDS gel and photographed under UV light. Any unincorporated HiLyte Fluor™ 488 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™ 488 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 HiLyte Fluor™ 488 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.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at email@example.com
|Baron, Desiree M et al.||ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function.||Cell reports||2022||ISSN 2211--1247|
|Lee, Gloria et al.||Active cytoskeletal composites display emergent tunable contractility and restructuring||Soft Matter||2021||ISSN 1744-6848|
|Cheng, Xianrui et al.||Xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization||Journal of Visualized Experiments||2021||ISSN 1940-087X|
|Goldstein-Levitin, Alina et al.||Intracellular functions and motile properties of bi-directional kinesin-5 cin8 are regulated by neck linker docking||eLife||2021||ISSN 2050-084X|
|Farhadi, Leila et al.||Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network||Soft Matter||2020||ISSN 1744-6848|
|Aher, Amol et al.||CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation||Current Biology||2020||ISSN 1879-0445|
|Nolet, Felix E. et al.||Nuclei determine the spatial origin of mitotic waves||eLife||2020||ISSN 2050-084X|
|Rodríguez-García, Ruddi et al.||Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules||Current Biology||2020||ISSN 1879-0445|
|Chen, Keyu et al.||Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules||Journal of Cell Biology||2020||ISSN 1540-8140|
|Nakos, Konstantinos et al.||Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- And filament-dependent manner||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Nakos, Konstantinos et al.||Regulation of microtubule plus end dynamics by septin 9||Cytoskeleton||2019||ISSN 1949-3592|
|Guedes-Dias, Pedro et al.||Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse||Current Biology||2019||ISSN 0960-9822|
|Zhu, Yili et al.||An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein||Bio-Protocol||2018||ISSN 2331--8325|
|Melo, Esther et al.||HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model||EBioMedicine||2018||ISSN 2352-3964|
|Aher, Amol et al.||CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain||Developmental Cell||2018||ISSN 1878-1551|
|Colin, Alexandra et al.||Actin-Network Architecture Regulates Microtubule Dynamics||Current Biology||2018||ISSN 0960-9822|
|McClintock, Mark A. et al.||RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs||eLife||2018||ISSN 2050-084X|
|Fan, Yuanwei et al.||The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus Ends||Current Biology||2018||ISSN 0960-9822|
|Arellano-Santoyo, Hugo et al.||A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity||Developmental Cell||2017||ISSN 1878-1551|
|Kandel, Mikhail E. et al.||Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy||ACS Nano||2017||ISSN 1936-086X|
|Zhang, Rui et al.||Interplay of structure, elasticity, and dynamics in actin-based nematic materials||Proceedings of the National Academy of Sciences of the United States of America||2017||ISSN 1091-6490|
|Britto, Mishan et al.||Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism||Proceedings of the National Academy of Sciences of the United States of America||2016||ISSN 1091-6490|
|Taberner, Núria et al.||Reconstituting Functional Microtubule-Barrier Interactions||Methods in Cell Biology||2014||ISSN 0091-679X|
|DeBerg, Hannah A. et al.||Motor domain phosphorylation modulates kinesin-1 transport||Journal of Biological Chemistry||2013||ISSN 0021-9258|
|Rostovtseva, Tatiana K. et al.||Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel *||Journal of Biological Chemistry||2012||ISSN 0021--9258|
|Hara, Masatoshi et al.||Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor||Nature Communications||2012||ISSN 2041-1723|
Question 1: Can HiLyte Fluor™ 488-labeled tubulin (Cat. # TL488M) be used to monitor tubulin dynamics in living cells?
Answer 1: Yes, all of Cytoskeleton’s fluorescently-labeled tubulins, including HiLyte Fluor™ 488-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. # TL488M 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™ 488-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.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org