Actin protein (rhodamine): rabbit skeletal muscle

Actin protein (rhodamine): rabbit skeletal muscle
$0.00

Product Uses Include

  • In vivo actin polymerization studies (microinjection into muscle cells)
  • In vitro motility studies using fluorescent F-actin and muscle myosins

Material
Purified rabbit muscle actin (Cat. # AKL99) has been modified to contain covalently linked rhodamine at random surface lysine residues. An activated ester of rhodamine is used to label the protein. The labeling stoichiometry has been determined to be 1-2 dyes per actin monomer. Rhodamine labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a pink lyophilized powder. The lyophilized protein is stable for 6 months when stored desiccated to <10% humidity at 4°C. The protein should be reconstituted to 10 mg/ml with distilled water, it will then be in the following buffer: 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 5% sucrose, and 1% dextran.

Rhodamine actin from a non-muscle source is also available (Cat. # APHR).

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. AR05 rhodamine muscle actin was found to be >99% pure (see Figure 1).

ar05gel

Figure 1. A 100 µg sample of rhodamine muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue. Protein quantitation was performed with the Precision Red Protein Assay Reagent (Cat. # ADV02).

Biological activity
The biological activity of rhodamine muscle actin can be 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 polymerized in this assay, which is similar to the unlabeled product (Cat. # AKL99)

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Krey, Jocelyn F. et al.Control of stereocilia length during development of hair bundlesPLOS Biology2023ISSN 1545--7885
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
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

 

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