Acti-stain 555 phalloidin

Acti-stain 555 phalloidin
$0.00

Product Uses Include

    • Fluorescent staining of actin filaments in fixed tissue sections and tissue culture cells preparations.Note: Unlike many actin antibodies, Acti-stain™ 555 phalloidin binds only to F-actin resulting in low background fluorescence.  Furthermore, actin staining is not appreciably different between species.
    • Preparation of stabilized fluorescent actin filaments in vitro.

       

      Actin staining is very useful in determining the structure and function of the cytoskeleton in living a nd fixed cells. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed with paraformaldehyde prior to probing or staining for actin structures. 

       

      Material

      Phalloidin is a seven amino acid peptide toxin from the mushroom Amanita phalloides, which binds specifically and with high affinity (Kd 20 nM) to the polymerized form of actin (F-actin).  Phalloidin lowers the critical concentration of actin polymerization to less than 1 µg/ml, thereby acting as a polymerization enhancer. Phalloidin has been labeled with a proprietary red fluorescent dye which allows it to be used to stain actin filaments in tissue cultured cells and tissue sections (1, see Figure 1) and cell-free preparations.  Acti-stain™ 555 phalloidin-labeled actin filaments retain many functional characteristics of unlabeled actin including their ability to interact with myosin.  Actin-stain™ 555 phalloidin is supplied as a red solid. 

      Note: Phalloidin is toxic and must be handled with care (LD50 human = 2mg/Kg).

       


       

      Example Results and Specifications

      Figure 1.   Actin Stress Fibers stained with Acti-stain™ 555 in a Swiss 3T3 cell.


      PHDH1_img1

      Swiss 3T3 cell with activated Rho, nucleus is stained with Dapi (blue) and F-actin is stained with Acti-stain™ 555 (red F-actin, Cat.# PHDH1).


      Figure 2. Emission and excitation scans for Acti-stain™ phalloidins


      PHDH1_scan

      Absorbance and fluorescence scan of Acti-stain™ 555. Labeled phalloidin was diluted into methanol and its absorbance and excitation spectra were scanned between 300-750 and 560-750 nm, respectively. Absorbance peaks at 560 nm and fluorescence at 575 nm.


      Acti-stain phalloidins are the most well characterized phalloidins available. Tabe 1 describes their brightness, photostability, background and affinity constants to F-actin. Compare these performance characteristics to other fluorescent phalloidins and you will see the advantages of using Acti-stain™ for your actin staining requirements.

      Table 1. Biochemical characteristics of fluorescent phalloidins

      ConjugateCat.#

      Wavelengths (Ex/Em)

      Brightness (AFU*)

      Stability to photobleaching(half life in seconds**)

      Background (% of total AFU at 100nM**)

      Affinity (Kd in nanomolar)

      Fluorescein-phalloidin

      na485/535 FITC filter set43262272 +/-12

      Acti-stain™ 488

      PHDG1485/535 FITC filter set83257555 +/-8

      Acti-stain™ 535

      PHDR1535/585 TRITC filter set430271236 +/-7

      Acti-stain™ 555

      PHDH1535/585 TRITC filter set551461663 +/-8

      Acti-stain™ 670

      PHDN1640/680 Cy5 filter set33281850 +/-12

      * = AFU's measured by quantitative cell imaging. ** = Measured in stained Swiss 3T3 cells in the absence of antifade.

       

      For more information about protocols to fix and stain cells click here.

       

      References

      1. Wulf, E. et al. (1979). Proc Natl Acad Sci USA. 76(9):4498-4502.

      2. Kron, S.J. et al. (1991).  Meth. Enzmol. 196: 399-416.

      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

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      Suzuki, Soichiro et al.ZNF185 prevents stress fiber formation through the inhibition of RhoA in endothelial cellsCommunications Biology 2023 6:12023ISSN 2399--3642
      Shoji, Kenji F et al.The mechanosensitive TRPV2 calcium channel promotes human melanoma invasiveness and metastatic potentialEMBO reports2023ISSN 1469--3178
      Karra, N. et al.The effect of membrane properties on cell growth in an ‘Airway barrier on a chip’Organs-on-a-Chip2023
      Bioactivity, MPF et al.Bioactivity Enhancement of Plasma-Sprayed Hydroxyapatite Coatings through Non-Contact Corona Electrical ChargingNanomaterials 2023, Vol. 13, Page 10582023ISSN 2079--4991
      Haines, A. et al.Chlamydia trachomatis Subverts Alpha-Actinins To Stabilize Its InclusionMicrobiology Spectrum2023ISSN 2165-0497
      Sohn, Claira et al.Pathogenic tau decreases nuclear tension in cultured neuronsFrontiers in Aging2023PMID 36756194
      O’Shea, T. M. et al.Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype in miceNature Communications 2022 13:12022ISSN 2041--1723
      Jain, Ishita et al.Delineating cooperative effects of Notch and biomechanical signals on patterned liver differentiationCommunications Biology 2022 5:12022ISSN 2399--3642
      Izumi, Hideki et al.Asymmetric Pericentrosomal CD133 Endosomes Induce the Unequal Autophagic Activity During Cytokinesis in CD133-Positive Human Neuroblastoma CellsStem Cells2022ISSN 1066--5099
      Cao, Yu Fei et al.Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinomaCell Death & Differentiation 2022 30:22022ISSN 1476--5403
      Huang, Qichao et al.TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasisThe EMBO Journal2022ISSN 1460--2075
      Music, Amna et al.The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentationFrontiers in Cell and Developmental Biology2022
      Winston, Tackla S. et al.Controlling Mesenchyme Tissue Remodeling via Spatial Arrangement of Mechanical ConstraintsFrontiers in Bioengineering and Biotechnology2022ISSN 2296-4185
      Küey, Cansu et al.Recruitment of clathrin to intracellular membranes is sufficient for vesicle formationeLife2022
      Sundby, Lauren J. et al.Nucleotide- and Protein-Dependent Functions of Actg1Molecular Biology of the Cell2022ISSN 1939-4586
      Hiratsuka, Ken et al.Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discoveryScience Advances2022ISSN 2375-2548
      Du, Qiqiao et al.FASN promotes lymph node metastasis in cervical cancer via cholesterol reprogramming and lymphangiogenesisCell Death & Disease 2022 13:52022ISSN 2041--4889
      Gupta, Dhanak et al.Traditional Multiwell Plates and Petri Dishes Limit the Evaluation of the Effects of Ultrasound on Cells In VitroUltrasound in Medicine & Biology2022
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      Zheng, Lijun et al.Ionizing radiation-induced long noncoding RNA CRYBG3 regulates YAP/TAZ through mechanotransductionCell Death & Disease 2022 13:32022ISSN 2041--4889
      Wang, Ling et al.Novel oral edaravone attenuates diastolic dysfunction of diabetic cardiomyopathy by activating the Nrf2 signaling pathwayEuropean Journal of Pharmacology2022ISSN 1879-0712
      Huang, Qichao et al.TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasisThe EMBO Journal2022ISSN 0261--4189
      Goering, Jeremy P. et al.In-frame deletion of SPECC1L microtubule association domain results in gain-of-function phenotypes affecting embryonic tissue movement and fusion eventsHuman Molecular Genetics2021ISSN 0964--6906
      Nanamatsu, Azuma et al.Vasopressin Induces Urinary Uromodulin Secretion by Activating PKA (Protein Kinase A)Hypertension2021ISSN 1524-4563
      Kimura, Ken ichi et al.Crucial role of framework with cytoskeletal actin filaments for shaping microstructure of footpad setae in the ladybird beetle, Harmonia axyridisArthropod Structure and Development2021ISSN 1873-5495
      Shindo, Taiki et al.Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transferJournal of biochemistry2021ISSN 1756-2651
      Gharanei, Seley et al.Vascular Adhesion Protein-1 Determines the Cellular Properties of Endometrial PericytesFrontiers in Cell and Developmental Biology2021ISSN 2296-634X
      Kanamoto, Takashi et al.Integrin α2β1 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gelScientific Reports2021ISSN 2045-2322
      Liang, Fei et al.Sorbs2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome patientseLife2021ISSN 2050-084X
      Liang, Wei et al.Necroptosis activates UPR sensors without disrupting their binding with GRP78Proceedings of the National Academy of Sciences of the United States of America2021ISSN 1091-6490
      Woo, Marcel S. et al.Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammationJournal of Experimental Medicine2021ISSN 1540-9538
      Schimmel, Lilian et al.Endothelial cells are not productively infected by SARS-CoV-2Clinical and Translational Immunology2021ISSN 2050-0068
      Goering, Jeremy P. et al.In-frame deletion of SPECC1L microtubule association domain results in gain-of-function phenotypes affecting embryonic tissue movement and fusion eventsHuman molecular genetics2021ISSN 1460-2083
      Schiweck, Juliane et al.Drebrin controls scar formation and astrocyte reactivity upon traumatic brain injury by regulating membrane traffickingNature Communications2021ISSN 2041-1723
      Wurzer, Hannah et al.Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome In Vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton RemodelingFrontiers in Immunology2021ISSN 1664-3224
      Haines, Adam et al.Cross Talk between ARF1 and RhoA Coordinates the Formation of Cytoskeletal Scaffolds during Chlamydia InfectionmBio2021ISSN 2150-7511
      Chen, Lena et al.Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line2020PMID 31965814
      Chemla, Yoav et al.Carbon nanostructures as a scaffold for human embryonic stem cell differentiation toward photoreceptor precursorsNanoscale2020ISSN 2040--3372
      Liu, Yi et al.Effect of F-actin and Microtubules on Cellular Mechanical Behavior Studied Using Atomic Force Microscope and an Image Recognition-Based Cytoskeleton Quantification ApproachInternational Journal of Molecular Sciences2020ISSN 1422-0067
      Hamdan, Hamdan et al.Mapping axon initial segment structure and function by multiplexed proximity biotinylationNature Communications2020ISSN 2041-1723
      Ding, Dalian et al.Some Ototoxic Drugs Destroy Cochlear Support Cells Before Damaging Sensory Hair CellsNeurotoxicity Research2020ISSN 1476-3524
      Pęziński, Marcin et al.An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machineryScientific Reports2020ISSN 2045-2322
      Hall, Everett G. et al.SPECC1L regulates palate development downstream of IRF6Human Molecular Genetics2020ISSN 1460-2083
      Ding, Dalian et al.Antioxidative stress-induced damage in cochlear explantsJournal of Otology2020ISSN 1672-2930
      Fu, Yi et al.Elevation of JAML Promotes Diabetic Kidney Disease by Modulating Podocyte Lipid MetabolismCell Metabolism2020ISSN 1932-7420
      Bokelmann, Marcel et al.Utility of primary cells to examine npc1 receptor expression in mops condylurus, a potential ebola virus reservoirPLoS Neglected Tropical Diseases2020ISSN 1935-2735
      Campisi, Marco et al.Tumor-Derived cGAMP Regulates Activation of the VasculatureFrontiers in Immunology2020ISSN 1664-3224
      Kimura, Ken ichi et al.Framework with cytoskeletal actin filaments forming insect footpad hairs inspires biomimetic adhesive device designCommunications Biology2020ISSN 2399-3642
      Sancey, Lucie et al.Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cellsJournal of Nanobiotechnology2020ISSN 1477-3155
      Shi, Rui et al.Rapid Osseointegration Bestowed by Carbonate Apatite Coating of Rough TitaniumAdvanced Materials Interfaces2020ISSN 2196-7350
      Chen, Lixia et al.CSRP2 suppresses colorectal cancer progression via p130Cas/Rac1 axis-meditated ERK, PAK, and HIPPO signaling pathwaysTheranostics2020ISSN 1838-7640
      Ueta, Miho et al.Effects of TGF-β1 on the migration and morphology of RAW264.7 cells in vitroMolecular Medicine Reports2019ISSN 1791-3004
      Ye, Haibo et al.Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic culturesNeuroToxicology2019ISSN 1872-9711
      Fultz, Robert et al.Phagocytosis by macrophages depends on histamine H2 receptor signaling and scavenger receptor 1MicrobiologyOpen2019ISSN 2045-8827
      Mandigo, Torrey R. et al.Drosophila emerins control LINC complex localization and transcription to regulate myonuclear positionJournal of Cell Science2019ISSN 1477-9137
      Ferguson, Sylvia H. et al.Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum InfectionCmgh2019ISSN 2352-345X
      Siddiqui, Nida et al.PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular transportNature Communications2019ISSN 2041-1723
      Vohnoutka, Rishel B. et al.The focal adhesion scaffold protein Hic-5 regulates vimentin organization in fibroblastsMolecular Biology of the Cell2019ISSN 1939-4586
      Ramos, Ana Raquel et al.Lipid phosphatases SKIP and SHIP2 regulate fibronectin-dependent cell migration in glioblastomaFEBS Journal2019ISSN 1742-4658
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      Del Signore, Steven J. et al.The WAVE Regulatory Complex and Branched F-Actin Counterbalance Contractile Force to Control Cell Shape and Packing in the Drosophila EyeDevelopmental Cell2018ISSN 1878-1551
      Bardai, Farah H. et al.A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivoJournal of Neuroscience2018ISSN 1529-2401
      Wang, Liqun et al.Tissue and cellular rigidity and mechanosensitive signaling activation in Alexander diseaseNature Communications2018ISSN 2041-1723
      Camuglia, Jaclyn M. et al.An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structureSkeletal Muscle2018ISSN 2044-5040
      Ghosh, Somadri et al.Inhibition of SHIP2 activity inhibits cell migration and could prevent metastasis in breast cancer cellsJournal of Cell Science2018ISSN 1477-9137
      Brauer, Philip R. et al.Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activityCell Communication and Adhesion2018ISSN 1543-5180
      Bardai, Farah H. et al.Lrrk promotes tau neurotoxicity through dysregulation of actin and mitochondrial dynamicsPLoS Biology2018ISSN 1545-7885
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      Della Torre, Camilla et al.Adsorption of B(α)P on carbon nanopowder affects accumulation and toxicity in zebrafish (Danio rerio) embryosEnvironmental Science: Nano2017ISSN 2051-8161
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      Dorland, Yvonne L. et al.The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctionsNature Communications2016ISSN 2041-1723
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      Question 1:  Can I use fluorescently-labeled phalloidin to stain F-actin in living cells?

      Answer 1:  Unfortunately, no, phalloidin cannot be used to stain F-actin in living cells.  Phalloidins are used to stain F-actin in fixed cells.  Fluorescent phalloidins only bind to the native quaternary structure of F-actin which provides a low background.  The correct fixation condition for phalloidin binding is 3.7% (v/v) paraformaldehyde in PBS for 10 min because it retains the quaternary protein structure which is necessary for high affinity binding of phalloidin.  Methanol fixation destroys the native conformation and hence is not suitable for F-actin staining with phalloidin.  To monitor actin dynamics in living cells, micro-injection of rhodamine-labeled actin (Cat. # APHR or AR05) is recommended.  Please see those datasheets for more information.

       

      Question 2:  Which of the fluorescently-labeled phalloidin is the most stable/brightest?

      Answer 2:  The brightest and most stable of the Acti-stains is Acti-stain 488 (green fluorescence; Cat. # PHDG1).  Please see the table below for additional information on all of our Acti-stains. 

      Conjugate

      Cat. #

      Wavelengths (Ex/EM)

      Brightness (AFU)

      Stability to photobleaching* (1/2 life in sec)

      Background (% of total AFU at 100 nM)

      Acti-stain™ 488

      PHDG1

      485/535

      832

      57

      5

      Acti-stain™ 535

      PHDR1

      535/585

      430

      27

      12

      Acti-stain™ 555

      PHDH1

      535/585

      551

      46

      16

      Acti-stain™ 670

      PHDN1

      640/680

      332

      18

      18

      * = measured in the absence of antifade.

       

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