Actin: HiLyte™ Fluor 555 Labeled (Rabbit skeletal muscle, >99% pure)

Actin: HiLyteFluor™ 555 Labeled (Rabbit skeletal muscle, >99% pure)

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Material

Purified rabbit muscle actin has been modified to contain covalently linked HiLyte™555 fluorochrome at random surface lysine residues. An activated ester of HiLyte™555 is used to label the protein. The labeling stoichiometry has been determined to be 0.8 – 1.4 dyes per actin monomer. HiLyte™555 labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a lyophilized powder (dark pink color). AR07 has maximal absorbance at 550 nm and emission at 570 nm (Fig. 1). See Application Table below for a variety of in vivo & in vitro uses for this reagent. 

 

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. HiLyte™555 labeled actin was found to be >99% pure (see Figure 2).

Applications

  Application  Reference
Modeling in vitro bio membranes  1, 2
Molecular Mechanisms underlying skeletal mediated force/stress  3, 4, 5, 6
in vitro modeling of the cytoskeleton in the cell cortex  7
Study mechanisms of in vivo actin dynamics by labeling of free barbed ends of actin filaments  8, 9, 10, 11
Study actin binding proteins  12, 13, 14
Applications in functional nanodevices  15, 16

 

Figure 1: Absorbance & Fluorescence Scan for AR07

ar07_-_fig1

Legend-Fig1: AR07 was diluted with nanopure water and its absorbance (green line) and fluorescence (orange line) spectra were scanned between 350 and 750 nm.  Fluorescent labeling stoichiometry was calculated to be 0.8-1.4 dyes per actin protein using the absorbance maximum for HiLyte™555 fluorescence at 540 nm and the Beer-Lambert law. The extinction coefficient of the dye is 150,000 M-1cm-1.

 

Figure 2: Actin HiLyte555 Protein Purity Determination

ar07_-_fig2

Legend-Fig. 2:  20 µg (Lanes 1 & 3) and 10 µg  (Lanes 2 & 4) of AR07 was analyzed by electrophoresis in a 4-20% SDS-PAGE system.  A Licor Odessy gel analysis was performed 600nm (HiLyte™555, lanes 1 & 2) and at 700nm (Coomassie, lanes 3 & 4), Protein quantitation was determined with the Precision Red™ Protein Assay Reagent (Cat. # ADV02).  Mark12 molecular weight markers are from Invitrogen.

Quality Control: Polymerization spin down assay
The biological activity of HiLyte™555 actin is determined by its ability to efficiently polymerize into filaments in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that ≥90% of the labeled muscle actin can polymerize in the presence of polymerization buffer & ≤5% poly-mer is present in the absence of polymerization buffer.

In vitro polymerization of HiLyte™555 actin to create labeled actin filaments

  1. Resuspend HiLyte™555 muscle actin to 0.4 mg/ml with General Actin Buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2; Cat. # BSA01) supplemented with 0.2 mM ATP and 1 mM DTT. 
  2. Add 1/10th the volume of  Polymerization Buffer (500 mM KCl, 20 mM MgCl2, 10 mM ATP; Cat. # BSA02) supplemented with 1 mM DTT and incubate at room temperature for 1 h.
  3. Dilute the polymerized actin filaments 100 fold in 1x Polymerization Buffer containing 70 nM phalloidin and spot 1 µl into a drop of anti-fade solution on a microscope slide.
  4. Place a coverslip over the drop and remove excess liquid with a tissue.
  5. Observe HiLyte™555 labeled actin filaments with a fluorescent microscope.
  6. A typical fluorescent image is shows in Figure 3.              

 

Figure 3: Fluorescent image of HiLyte™555 actin filaments

ar07_-_fig3

HiLyte™555 actin  muscle actin was polymerized for 1 h, spotted onto a microscope slide and observed by epi-fluorescence microscopy equipped with a digital CCD camera and 100x objective. Fluorescent filaments were observed using a TRITC filter set Ex: 525±15 / Em: 595±20

 

Application References

1-  Design and construction of a multi-tiered minimal actin cortex    for structural support in lipid bilayer applications. 2024. Smith A.J. et al. Appl. Bio. Mater. 7: 1936-1946

2-   In vitro reconstruction of the actin cytoskeleton inside giant unilamellar vesicles. 2022. Chen S. et al. Jove J. 10.3791/64026

3-   Reconstituting and characterizing actin-microtubule composites with tunable motor driven dynamics and mechanics. 2022. Sasanpour M. et al. Jove J. 10.3791/64228

4-   Molecular mechanism for direct actin force-sensing by alpha-catenin. 2020. Mei L. et al. eLife 9:e62514

5-   Anillin propels myosin-independent constriction of actin rings. 2021. Kucera O. et al. Nature Comm. 10.1038/s41467-021-24474-1

6-   Bending forces and nucleotide state jointly regulate F-actin structure. 2022. Reynolds M. et al. Nature 611: 380-386

7-   Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex. 2022. Wu H. et al. PNAS 119: 10 e2115217119

8-   Control of stereocilia length during development of hair bundles. 2023. Krey J.F. et al. PLOS Bio. 10.137/journal.pbio.3001964

9- Arp2/3 and Mena/VASP require profilin 1 for actin network assembly at the leading edge. 2020. Skruber K. et al. Curr. Bio. 30: 2651-2664

10- Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin. 2021. McGrath J. et al. Curr. Bio. 31:1141-1153

11- EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells. 1998. Chen A.Y. et al. J. Cell Sci. 111: 199-211

12- Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity. 2021. Cell 184: 4016-4031

13- Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin. 2024. Morales E.A. et al. J. Biol. Chem. 300(5) 107279

14- Dynamin-2 regulates postsynaptic cytoskeleton organization and neuromuscular junction development. 2020. Lin S. et al. Cell Rep. 33: 108310

15- Comparison of actin-and microtubule-based motility systems for application in functional nanodevices. 2021. Reuther C. et al. New J. Phys. 23:075007

16- The potential of myosin and actin in nanobiotechnology. 2023. Mansson A. J. Cell Sci. 136: 10.1242/jcs.261025

 

For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Arslan, Feyza Nur et al.Adhesion-induced cortical flows pattern E-cadherin-mediated cell contactsCurrent biology : CB2024ISSN 1879--0445
Sakamoto, Ryota et al.F-actin architecture determines the conversion of chemical energy into mechanical workNature Communications 2024 15:12024ISSN 2041--1723
Morales, E. Angelo et al.Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actinJournal of Biological Chemistry2024ISSN 0021--9258
Smith, Amanda J. et al.Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer ApplicationsACS Applied Bio Materials2024ISSN 2576-6422
Månsson, AlfThe potential of myosin and actin in nanobiotechnologyJournal of Cell Science2023ISSN 1477-9137
Krey, Jocelyn F. et al.Control of stereocilia length during development of hair bundlesPLOS Biology2023ISSN 1545--7885
Wu, Huayin et al.Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortexProceedings of the National Academy of Sciences of the United States of America2022ISSN 1091-6490
Chen, Sheng et al.In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar VesiclesJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Henty-Ridilla, Jessica L.Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) MicroscopyJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Reynolds, Matthew J. et al.Bending forces and nucleotide state jointly regulate F-actin structureNature 2022 611:79352022ISSN 1476--4687
Sasanpour, Mehrzad et al.Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and MechanicsJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Reuther, Cordula et al.Comparison of actin- and microtubule-based motility systems for application in functional nanodevicesNew Journal of Physics2021ISSN 1367--2630
Giampazolias, Evangelos et al.Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunityCell2021ISSN 1097-4172
McGrath, Jamis et al.Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilinCurrent Biology2021ISSN 1879-0445
Kučera, Ondřej et al.Anillin propels myosin-independent constriction of actin ringsNature Communications2021ISSN 2041-1723
Lin, Shan Shan et al.Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction DevelopmentCell Reports2020ISSN 2211-1247
Skruber, Kristen et al.Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading EdgeCurrent Biology2020ISSN 1879-0445
Sun, Xiaoyu et al.Mechanosensing through Direct Binding of Tensed F-Actin by LIM DomainsDevelopmental Cell2020ISSN 1878-1551
Padmanabhan, Krishnanand et al.Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarityDevelopment (Cambridge)2020ISSN 1477-9129
Farhadi, Leila et al.Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal networkSoft Matter2020ISSN 1744-6848
Mei, Lin et al.Molecular mechanism for direct actin force-sensing by α-catenineLife2020ISSN 2050-084X
Chan, Byron et al.Adseverin, an actin binding protein, regulates articular chondrocyte phenotypeJournal of Tissue Engineering and Regenerative Medicine2019ISSN 1932-7005
Beutel, Oliver et al.Phase Separation of Zonula Occludens Proteins Drives Formation of Tight JunctionsCell2019ISSN 1097-4172
Zeng, Menglong et al.Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and PlasticityCell2018ISSN 1097-4172
Burden, Daniel L. et al.Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin CortexLangmuir2018ISSN 1520-5827
Silván, Unai et al.Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopyJournal of Structural Biology2016ISSN 1095-8657
Jiang, Hongwei et al.Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone lossFASEB Journal2015ISSN 1530-6860
Ramamurthy, Bhagavathi et al.Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VICytoskeleton2012ISSN 1949-3584
Del Duca, Stefano et al.Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubulesand actin filamentsBiochemical Journal2009ISSN 1470-8728
Klaavuniemi, Tuula et al.Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeatsJournal of Biological Chemistry2008ISSN 0021-9258
Chan, Amanda Y. et al.EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cellsJournal of Cell Science1998ISSN 0021--9533

 

Question 1:  What is the best way to store actin proteins to insure maximum stability and shelf-life?

Answer 1:  Cytoskeleton provides all actin proteins as lyophilized powders so that they can be shipped at room temperature.  Upon receipt, the lyophilized powders should be stored at 4°C in a sealed container with desiccant.  It is important to monitor the freshness of the desiccant and insure that it continues to absorb moisture to protect the lyophilized actins.  With proper storage, the lyophilized actins are guaranteed to be stable for 6 months.  Alternatively, actins can be immediately resuspended at the concentration recommended, aliquoted, snap-frozen in liquid nitrogen and stored at -70°C.  When thawing frozen aliquots, it is important to thaw rapidly in a room temperature water bath.

 

Question 2:  What is the best way to store F-actin after polymerizing?

Answer 2:  G-actin is stable for two days at 4°C and requires a divalent cation, pH 6.5 - 8.0 and ATP for stability.  F-actin is stable and can be stored at 4°C for 1-2 weeks.  F-actin requires ATP (0.2 mM) and Mg2+ (2 mM) for stability and is unstable below pH 6.5 and above pH 8.5.  F-actin is not stable to freezing.  F-actin can be transferred to a variety of buffers (e.g. HEPES, phosphate, etc) without detrimental effects.  We recommend the addition of antibacterial agents such as 100 μg/ml ampicillin and 10 μg/ml chloramphenicol when storing F-actin at 4°C.

 

Question 3: Filters for visualizing HiLyte™ 555 signal?

Answer 3:  The excitation filter should be set at 525 nm and the emission filter at 595 nm.

 

 

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com