Actin - HiLyte Fluor 555 Labeled (Rabbit skeletal muscle - >99% pure)
Actin - HiLyte Fluor 555 Labeled (Rabbit skeletal muscle - >99% pure) (AR07-GRP)
Products
Description
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
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 HiLyte™555 Protein Purity Determination
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
- 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.
- 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.
- 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.
- Place a coverslip over the drop and remove excess liquid with a tissue.
- Observe HiLyte™555 labeled actin filaments with a fluorescent microscope.
- A typical fluorescent image is shows in Figure 3.
Figure 3: Fluorescent image of HiLyte™555 actin filaments
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
Specifications
| Storage | 4°C |
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