Actin protein (rhodamine): rabbit skeletal muscle

Actin protein (rhodamine): rabbit skeletal muscle
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Material

Purified rabbit muscle actin has been modified to contain covalently linked rhodamine fluorochrome at random surface lysine residues. An activated ester of  rhodamine is used to label the protein. The labeling stoichiometry has been determined to be 0.8 – 1.4 dyes per actin monomer. Rhodamine labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a lyophilized powder (dark pink color). AR05 has maximal absorbance at 545 nm and emission at 585 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.  Rhodamine 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 AR05

ar05_-_fig1

Legend-Fig. 1: AR05 was diluted with nanopure water and its absorbance (green line) and fluorescence (red line) spectra were scanned between 300 and 750 nm.  Fluorescent labeling stoichiometry was calculated to be 0.8-1.4 dyes per actin protein using the absorbance maximum for  rhodamine actin at 545 nm and the Beer-Lambert law. The extinction coefficient of the dye is 85,800 M-1cm-1.

 

 

Figure 2: Actin Rhodamine Protein Purity Determination

ar05_-_fig2

Legend-Fig. 2:  20 µg (Lanes 1 & 3) and 10 µg  (Lanes 2 & 4) of AR05 was analyzed by electrophoresis in a 4-20% SDS-PAGE system.  A Licor Odessy gel analysis was performed 600nm (Rhodamine, 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 rhodamine 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 rhodamine actin to create labeled actin filaments

  1. Resuspend rhodamine 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 rhodamine labeled actin filaments with a fluorescent microscope.
  6. A typical fluorescent image is shows in Figure 3.              

 

Figure 3: Fluorescent image of rhodamine actin filaments

ar05_-_fig3

Rhodamine actin was polymerized for 1 h, spotted onto a microscope slide and observed by epifluorescence 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
Sakamoto, Ryota et al.Composite branched and linear F-actin maximize myosin-induced membrane shape changes in a biomimetic cell modelCommunications Biology2024
Sakamoto, R. et al.Active tension and membrane friction mediate cortical flows and blebbing in a model actomyosin cortexPhysical Review Research2024
Arslan, Feyza Nur et al.Adhesion-induced cortical flows pattern E-cadherin-mediated cell contactsCurrent biology : CB2024
Sakamoto, Ryota et al.F-actin architecture determines the conversion of chemical energy into mechanical workNature Communications 2024
Morales, E. Angelo et al.Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actinJournal of Biological Chemistry2024
Smith, Amanda J. et al.Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer ApplicationsACS Applied Bio Materials2024
Okura, Kaoru et al.Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin FilamentsJournal of Molecular Biology2023
Chen, Xudong et al.Phase separation-mediated actin bundling by the postsynaptic density condensateseLife2023
Wang, Yanan et al.PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell ComplexNature Communications2023
Yu, Yiming et al.Self-assembly of CIP4 drives actin-mediated asymmetric pit-closing in clathrin-mediated endocytosisNature Communications2023
Nietmann, Peter et al.Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological functionNature Communications2023
Månsson, AlfThe potential of myosin and actin in nanobiotechnologyJournal of Cell Science2023
Krey, Jocelyn F. et al.Control of stereocilia length during development of hair bundlesPLOS Biology2023
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 America2022
Chen, Sheng et al.In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar VesiclesJoVE (Journal of Visualized Experiments)2022
Henty-Ridilla, Jessica L.Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) MicroscopyJoVE (Journal of Visualized Experiments)2022
Reynolds, Matthew J. et al.Bending forces and nucleotide state jointly regulate F-actin structureNature 2022 611:79352022
Sasanpour, Mehrzad et al.Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and MechanicsJoVE (Journal of Visualized Experiments)2022
Reuther, Cordula et al.Comparison of actin- and microtubule-based motility systems for application in functional nanodevicesNew Journal of Physics2021
Giampazolias, Evangelos et al.Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunityCell2021
McGrath, Jamis et al.Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilinCurrent Biology2021
Kučera, Ondřej et al.Anillin propels myosin-independent constriction of actin ringsNature Communications2021
Lin, Shan Shan et al.Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction DevelopmentCell Reports2020
Skruber, Kristen et al.Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading EdgeCurrent Biology2020
Sun, Xiaoyu et al.Mechanosensing through Direct Binding of Tensed F-Actin by LIM DomainsDevelopmental Cell2020
Padmanabhan, Krishnanand et al.Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarityDevelopment (Cambridge)2020
Farhadi, Leila et al.Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal networkSoft Matter2020
Mei, Lin et al.Molecular mechanism for direct actin force-sensing by α-catenineLife2020
Chan, Byron et al.Adseverin, an actin binding protein, regulates articular chondrocyte phenotypeJournal of Tissue Engineering and Regenerative Medicine2019
Beutel, Oliver et al.Phase Separation of Zonula Occludens Proteins Drives Formation of Tight JunctionsCell2019
Zeng, Menglong et al.Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and PlasticityCell2018
Burden, Daniel L. et al.Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin CortexLangmuir2018
Silván, Unai et al.Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopyJournal of Structural Biology2016
Jiang, Hongwei et al.Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone lossFASEB Journal2015
Ramamurthy, Bhagavathi et al.Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VICytoskeleton2012
Del Duca, Stefano et al.Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubulesand actin filamentsBiochemical Journal2009
Klaavuniemi, Tuula et al.Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeatsJournal of Biological Chemistry2008
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 Science1998

 

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 rhodamine signal?

Answer 3:  The excitation filter should be set at 535 nm and the emission filter at 585 nm.

 

 

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