Actin protein (rhodamine): human platelet

Actin protein (rhodamine): human platelet
$0.00

Product Uses Include

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

Material
Non-muscle actin purified from human platelets (Cat. # APHL99) 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 0.4 dyes per actin monomer. Rhodamine non-muscle actin has an approximate molecular weight of 43 kDa. Rhodamine non-muscle actin is supplied as a pink lyophilized powder. The lyophilized protein is stable for 6 months wh en 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 muscle source is also available (Cat. # AR05).

Live CHO cell injected with rhodamine beta-actin.

CHO cell micro-injected with rhodamine non-muscle actin (Cat. # APHR). Dr. R. Goldman's Lab (Northwestern University, USA)

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. APHR rhodamine non-muscle actin is >99% pure (see Figure 1). No free dye is detectable in the final product

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Figure 1. Rhodamine Non-Muscle Actin Protein Purity Determination. A 100 µg sample of rhodamine non-muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue. Minor protein bands present at 86 and 120 kDa are dimers and trimers of rhodamine actin respectively, and constitute <1% of the total protein. Protein quantitation was performed with the Precision Red Protein Assay Reagent (Cat. # ADV02).

Biological activity
The biological activity of rhodamine non-muscle 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 >80% of the labeled non-muscle actin polymerizes in this assay, which is comparable to the unconjugated protein (APHL99)

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
Pollitt, Stephanie L. et al.LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon developmentMolecular Biology of the Cell2020ISSN 1939-4586
Biel, Natalie et al.Imaging intranuclear actin rods in live heat stressed drosophila embryosJournal of Visualized Experiments2020ISSN 1940-087X
Nakos, Konstantinos et al.Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- And filament-dependent mannerMolecular Biology of the Cell2019ISSN 1939-4586
Nikitina, E. A. et al.A Weakened Geomagnetic Field: Effects on Genomic Transcriptiln Activity, Learning, and Memory in Drosophila MelanogasterNeuroscience and Behavioral Physiology2018ISSN 1573-899X
Muriel, Olivia et al.In vitro polymerization of f-actin on early endosomesJournal of Visualized Experiments2017ISSN 1940-087X
DeBruhl, Heather et al.Rop, the Sec1/Munc18 homolog in Drosophila, is required for furrow ingression and stable cell shape during cytokinesisJournal of Cell Science2016ISSN 1477-9137
Delorme-Walker, Violaine et al.Chronophin coordinates cell leading edge dynamics by controlling active cofilin levelsProceedings of the National Academy of Sciences of the United States of America2015ISSN 1091-6490
Marsick, Bonnie M. et al.Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth conesJournal of Visualized Experiments2011ISSN 1940-087X
Castro, Javier S. et al.Binding affinity of fluorochromes and fluorescent proteins to Taxol™ crystalsMaterials Science and Engineering C2009ISSN 0928-4931
Gupton, Stephanie L. et al.Live-Cell Fluorescent Speckle Microscopy of Actin Cytoskeletal Dynamics and Their Perturbation by Drug PerfusionCell Biology, Four-Volume Set2006Article Link
Tsukada, Miki et al.Doublecortin Association with Actin Filaments Is Regulated by Neurabin II *Journal of Biological Chemistry2005ISSN 0021--9258
Vasanji, Amit et al.Polarization of Plasma Membrane Microviscosity during Endothelial Cell MigrationDevelopmental Cell2004ISSN 1534--5807
Idrissi, Fatima Zahra et al.Cofilin, but not profilin, is required for myosin-I-induced actin polymerization and the endocytic uptake in yeastMolecular Biology of the Cell2002ISSN 1059-1524
Smilenov, Lubomir B. et al.Focal adhesion motility revealed in stationary fibroblastsScience1999ISSN 0036-8075
Machesky, Laura M. et al.Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal ReorganizationJournal of Cell Biology1997ISSN 0021--9525

 

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, lyophilized actin is guaranteed to be stable for 6 months.  Alternatively, actin 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:  What are the appropriate filter settings 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