Actin: HiLyte™ Fluor 488 Labeled (Rabbit skeletal muscle, >99% pure)
Actin: HiLyteFluor™ 488 Labeled (Rabbit skeletal muscle, >99% pure)
Material
Purified rabbit muscle actin has been modified to contain covalently linked HiLyte™488 fluorochrome at random surface lysine residues. An activated ester of HiLyte™488 is used to label the protein. The labeling stoichiometry has been determined to be 0.8 – 1.4 dyes per actin monomer. HiLyte™488 labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a lyophilized powder (faint yellow/orange color). AR06 has maximal absorbance at 499 nm and emission at 523 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™488 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 AR06
Legend-Fig1: AR06 was diluted with nanopure water and its absorbance (blue line) and fluorescence (green 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™488 fluorescence at 523 nm and the Beer-Lambert law. The extinction coefficient of the dye is 70,000 M-1cm-1.
Figure 2: Actin HiLyte™488 Protein Purity Determination
Legend-Fig 2: 20 µg (Lanes 1 & 3) and 10 µg (Lanes 2 & 4) of AR06 were analyzed by electrophoresis in a 4-20% SDS-PAGE system. A Li-Cor Odessy gel analysis was performed 600 nm (HiLyte™488, lanes 1 & 2) and at 700 nm (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™488 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™488 actin to create labeled actin filaments
- Resuspend HiLyte™488 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™488 labeled actin filaments with a fluorescent microscope.
- A typical fluorescent image is shows in Figure 3.
Figure 3: Fluorescent image of HiLyte™488 actin filaments
HiLyte™488 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 FITC filter set Ab: 480±15 / Em: 535±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
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™ 488 signal?
Answer 3: The excitation filter should be set at 480 nm and the emission filter at 535 nm.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
