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.
|Planelles-Herrero, Vicente Jose et al.||Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants||Nature Cell Biology||2022||ISSN 1476--4679|
|Havelka, Daniel et al.||Lab-on-chip microscope platform for electro-manipulation of a dense microtubules network||Scientific Reports||2022||ISSN 2045-2322|
|Hochmair, Janine et al.||Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates||The EMBO Journal||2022||ISSN 0261--4189|
|Watson, Joseph L. et al.||High-efficacy subcellular micropatterning of proteins using fibrinogen anchors||Journal of Cell Biology||2021||ISSN 1540-8140|
|Saper, Gadiel et al.||Robotic end-to-end fusion of microtubules powered by kinesin||Science Robotics||2021||ISSN 2470-9476|
|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|
|Bassir Kazeruni, Neda M. et al.||Microtubule Detachment in Gliding Motility Assays Limits the Performance of Kinesin-Driven Molecular Shuttles||Langmuir||2020||ISSN 1520-5827|
|Tseng, Kuo Fu et al.||The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility||Current Biology||2020||ISSN 1879-0445|
|Gaska, Ignas et al.||The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule Sliding||Developmental Cell||2020||ISSN 1878-1551|
|Aher, Amol et al.||CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation||Current Biology||2020||ISSN 1879-0445|
|Zehr, Elena A. et al.||Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules||Developmental Cell||2020||ISSN 1878-1551|
|Diwaker, Drishya et al.||Deletion of the Pseudorabies virus gE/gI-US9p complex disrupts kinesin KIF1A and KIF5C recruitment during egress, and alters the properties of microtubule-dependent transport in vitro||PLoS Pathogens||2020||ISSN 1553-7374|
|von Appen, Alexander et al.||LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation||Nature||2020||ISSN 1476-4687|
|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|
|Rodríguez-García, Ruddi et al.||Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules||Current Biology||2020||ISSN 1879-0445|
|Li, Feiran et al.||Local direction change of surface gliding microtubules||Biotechnology and Bioengineering||2019||ISSN 1097-0290|
|Jiang, Shuo et al.||Interplay between the Kinesin and Tubulin Mechanochemical Cycles Underlies Microtubule Tip Tracking by the Non-motile Ciliary Kinesin Kif7||Developmental Cell||2019||ISSN 1878-1551|
|Siddiqui, Nida et al.||PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular transport||Nature Communications||2019||ISSN 2041-1723|
|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|
|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|
|Nakos, Konstantinos et al.||Regulation of microtubule plus end dynamics by septin 9||Cytoskeleton||2019||ISSN 1949-3592|
|Kazeruni, Neda M.Bassir et al.||Assembling molecular shuttles powered by reversibly attached kinesins||Journal of Visualized Experiments||2019||ISSN 1940-087X|
|Chen, Yang et al.||Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems||Current Protocols in Cell Biology||2018||ISSN 1934-2616|
|Pesenti, Marion E. et al.||Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80||Molecular Cell||2018||ISSN 1097-4164|
|Aher, Amol et al.||CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain||Developmental Cell||2018||ISSN 1878-1551|
|Wang, Pan et al.||The Central Stalk Determines the Motility of Mitotic Kinesin-14 Homodimers||Current Biology||2018||ISSN 0960-9822|
|Colin, Alexandra et al.||Actin-Network Architecture Regulates Microtubule Dynamics||Current Biology||2018||ISSN 0960-9822|
|Karasmanis, Eva P. et al.||Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))||Developmental Cell||2018||ISSN 1878-1551|
|McIntosh, Betsy B. et al.||Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal Networks||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|
|Gramlich, Michael W. et al.||Single molecule investigation of kinesin-1 motility using engineered microtubule defects||Scientific Reports||2017||ISSN 2045-2322|
|Maciejowski, John et al.||Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation||Developmental Cell||2017||ISSN 1878-1551|
|Zhang, Kai et al.||Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated||Cell||2017||ISSN 1097-4172|
|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|
|DeBruhl, Heather et al.||Rop, the Sec1/Munc18 homolog in Drosophila, is required for furrow ingression and stable cell shape during cytokinesis||Journal of Cell Science||2016||ISSN 1477-9137|
|Hara, Yuki et al.||Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts||Developmental Cell||2015||ISSN 1878-1551|
|Norris, Stephen R. et al.||Influence of fluorescent tag on the motility properties of kinesin-1 in single-molecule assays||Biophysical Journal||2015||ISSN 1542-0086|
|Dumont, Emmanuel L.P. et al.||Molecular wear of microtubules propelled by surface-adhered kinesins||Nature Nanotechnology 2015 10:2||2015||ISSN 1748--3395|
|Roth-Johnson, Elizabeth A. et al.||Interaction between microtubules and the drosophila formin cappuccino and its effect on actin assembly||Journal of Biological Chemistry||2014||ISSN 0021-9258|
|Leslie, Kris et al.||Going Solo: Measuring the motions of microtubules with an in vitro assay for tirf microscopy.||Methods in Cell Biology||2013||ISSN 0091-679X|
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.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org