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Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin

Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin
$0.00
SKU
BK003
Datasheet
MSDS
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Product Uses Include

  • To show quantitative / qualitative effects on actin polymerization by the addition of a extract, an actin binding protein or compound.
  • To show quantitative / qualitative effects on actin polymerization by addition of an F-actin nucleating protein, compound or extract.
  • To show quantitative / qualitative effects on steady-state F-actin levels by addition of an F-actin severing protein, compound or extract.
  • To show quantitative / qualitative effects on actin depolymerization by addition of an actin binding protein, compound or extract.

Introduction
The Actin Polymerization Biochem Kit™ is based on the enhanced fluorescence of pyrene conjugated actin that occurs during polymerization. The enhanced fluorescence that occurs when pyrene G-actin (monomer) forms pyrene F-actin can be measured in a fluorimeter to follow polymerization over time. Also, by using preformed pyrene F-actin, it is possible to follow depolymerization. Both cell/tissue extracts and purified proteins can be added to the reaction mixture to identify their effect on actin polymerization. The components of the kit can also be used separately for other actin based assays such as a spin-down assays to detect F-actin binding proteins (see also BK001) or size exclusion chromatography to identify G-actin binding proteins. See the About Actin page for more information on assays testing actin binding proteins.

While this kit comes with pyrene labeled skeletal muscle actin, it can also be used to study polymerization of other types of actin such as non-muscle actin (Cat. # APHL99) or cardiac actin (Cat. # AD99). Polymerization assays with these actins can be performed using a 10:1 ratio between the actin you want to study and the included pyrene actin

Kit contents
The kit contains enough materials for 30-100 assays depending on assay volume. The following reagents are included:

  1. 5 x 1 mg Pyrene labeled actin (Cat. # AP05). 
  2. General Actin Buffer (Cat. # BSA01).
  3. Actin Polymerization Buffer (Cat. # BSA02).
  4. ATP 100mM (Cat. # BSA04).
  5. Tris-HCl pH 7.5, 100 mM
  6. Manual with detailed protocols and extensive troubleshooting guide.

Equipment needed

  1. Fluorescence spectrophotometer (cuvette or 96-well plate) with 4-10 nm bandwidth at 365 nm excitation wavelength, and 4-10 nm bandwidth at 407 nm emission wavelength.
  2. Small capacity (100-1000 µl) fluorescence spectrophotometer cuvette or 96-well plate.

Example results
The Actin Polymerization Biochem Kit™ was used to study the effects of Arp2/3 (Cat. # RP01) and the VCA domain of WASP (Cat. # VCG03) on actin polymerization rates. The Arp2/3 complex is an actin filament nucleator but has low nucleating/polymerizing activity on its own. The VCA domain of WASP is an activator of the Arp2/3 complex. Hence, when the Arp2/3 complex is mixed with the WASP VCA domain, these two exert a potent actin polymerizing activity (Fig. 1).

Click Here for Detailed Methods
arp-actin-pol

Figure 1. Actin polymerization stimulated by Arp2/3 complex and the VCA domain of WASP. Actin polymerization was measured using kit BK003. The addition of Arp2/3 complex or the VCA domain alone to actin has minimal effects on actin polymerization, while the combination of Arp2/3 and the VCA domain strongly stimulates the rate of actin polymerization.

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

 

  For our Actin Polymerization Excel Template please download here. 

    AuthorTitleJournalYearArticle Link
    Lyu, Jialan et al.Cypher/ZASP drives cardiomyocyte maturation via actin-mediated MRTFA-SRF signallingTheranostics2024
    Akter, Tanjina et al.IQGAP1 Regulates Actin Polymerization and Contributes to Bleomycin-Induced Lung FibrosisInternational Journal of Molecular Sciences2024
    Huang, Xuejiao et al.Polyglutamine Binding Protein 1 regulates neurite outgrowth through recruiting N-WASPJournal of Biological Chemistry2024
    Kumar, Raj et al.Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular LeakageArteriosclerosis, Thrombosis, and Vascular Biology2024
    Formánek, Bedřich et al.Synthesis and migrastatic activity of cytochalasin analogues lacking a macrocyclic moietyRSC Medicinal Chemistry2024
    Chand, Abhishu et al.CdSe/ZnS Quantum Dots’ Impact on In Vitro Actin DynamicsInternational Journal of Molecular Sciences 2024
    Chanez-Paredes, Sandra D. et al.Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variantsJournal of Biological Chemistry2024
    Scanlon, Kaylyn R. et al.Chlamydia trachomatis TmeB antagonizes actin polymerization via direct interference with Arp2/3 activityFrontiers in Cellular and Infection Microbiology2023
    Coverdale, J. P.C. et al.Triplex metallohelices have enantiomer-dependent mechanisms of action in colon cancer cellsDalton Transactions2023
    Tian, Huayang et al.Phase separation of S-RNase promotes self-incompatibility in Petunia hybridaJournal of Integrative Plant Biology2023
    Zhang, Qing et al.Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavHPLOS Pathogens2023
    Bogucka-Janczi, Katarzyna et al.ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.eLife2023
    Ángeles-Robles, Gabriela et al.Actin protein inside DMPC GUVs and its mechanical response to AC electric fieldsBiochimica et Biophysica Acta (BBA) - Biomembranes2022
    Geng, Mengxin et al.Synthesis and characterization of semisynthetic analogs of the antifungal occidiofunginFrontiers in Microbiology2022
    Chen, Binghong et al.Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signalingJournal of Experimental and Clinical Cancer Research2022
    Wang, Baolei et al.Numb and Numblike regulate sarcomere assembly and maintenanceJournal of Clinical Investigation2022
    Hsiao, Bo Yuan et al.Human costars family protein abracl modulates actin dynamics and cell migration and associates with tumorigenic growthInternational Journal of Molecular Sciences2021
    Misra, Suniti et al.Periostin/Filamin-A: A Candidate Central Regulatory Axis for Valve Fibrogenesis and Matrix CompactionFrontiers in Cell and Developmental Biology2021
    Bock, Fabian et al.Rac1 promotes kidney collecting duct integrity by limiting actomyosin activityJournal of Cell Biology2021
    Yan, Yanling et al.RTKN-1/rhotekin shields endosome-associated F-actin from disassembly to ensure endocytic recyclingJournal of Cell Biology2021
    Sarkar, Souvarish et al.Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivoMolecular Neurodegeneration2021
    Shimura, Daisuke et al.Protective mitochondrial fission induced by stress-responsive protein GJA1-20keLife2021
    Chen, Yuewen et al.Coronin 2B regulates dendrite outgrowth by modulating actin dynamicsFEBS Letters2020
    Kang, Dong Hee et al.Down-regulation of diesel particulate matter-induced airway inflammation by the PDZ motif peptide of ZO-1Journal of Cellular and Molecular Medicine2020
    Lamm, Noa et al.Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stressNature Cell Biology2020
    Schneidewind, Tabea et al.The Pseudo Natural Product Myokinasib Is a Myosin Light Chain Kinase 1 Inhibitor with Unprecedented ChemotypeCell Chemical Biology2019
    Limatola, Nunzia et al.Nicotine Induces Polyspermy in Sea Urchin Eggs through a Non-Cholinergic Pathway Modulating Actin DynamicsCells2019
    Li, Linxi et al.Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organizationCell Death and Disease2019
    Liu, Juan et al.CCR7 Chemokine Receptor-Inducible lnc-Dpf3 Restrains Dendritic Cell Migration by Inhibiting HIF-1α-Mediated GlycolysisImmunity2019
    Li, Lin Xi et al.Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro studyAsian Journal of Andrology2019
    Lang, Lei et al.ATM-Mediated phosphorylation of cortactin involved in actin polymerization promotes breast cancer cells migration and invasionCellular Physiology and Biochemistry2019
    Zhang, Min et al.Axonogenesis Is Coordinated by Neuron-Specific Alternative Splicing Programming and Splicing Regulator PTBP2Neuron2019
    Ravichandran, Akshaya et al.A novel actin binding drug with in vivo efficacyAntimicrobial Agents and Chemotherapy2019
    Kommaddi, Reddy Peera et al.Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in alzheimer’s diseaseJournal of Neuroscience2018
    Wen, Qing et al.Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testisCell Death and Disease2018
    Bardai, Farah H. et al.Lrrk promotes tau neurotoxicity through dysregulation of actin and mitochondrial dynamicsPLoS Biology2018
    Gau, David et al.Structure-based virtual screening identifies a small-molecule inhibitor of the profilin 1–actin interactionJournal of Biological Chemistry2018
    Shioda, Norifumi et al.Targeting G-quadruplex DNA as cognitive function therapy for ATR-X syndrome articleNature Medicine2018
    Kim, Mi Seon et al.Src is the primary target of aripiprazole, an atypical antipsychotic drug, in its anti-tumor actionOncotarget2018
    Patel, Vaibhav B. et al.PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart diseaseNature Communications2018
    Kumar, Dhivya et al.Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzymeScientific Reports2018
    Sarowar, Tasnuva et al.Object phobia and altered RhoA signaling in amygdala of mice lacking RICH2Frontiers in Molecular Neuroscience2017
    Misiak, Majus et al.Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7Oncotarget2017
    Tormos, Ana M. et al.P38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and agingPLoS ONE2017
    Wu, Tongbin et al.HSPB7 is indispensable for heart development by modulating actin filament assemblyProceedings of the National Academy of Sciences of the United States of America2017
    Kumar, Raj et al.A new role for cofilin in retinal neovascularizationJournal of Cell Science2016
    Sarowar, Tasnuva et al.Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2Molecular Brain2016
    Kpetemey, Marilyne et al.MIEN1 drives breast tumor cell migration by regulating cytoskeletal-focal adhesion dynamicsOncotarget2016
    Rondina, M. T. et al.Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human plateletsJournal of Thrombosis and Haemostasis2016
    Chakraborty, Sayan et al.An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinomaNature Communications2015
    Jiang, Shiyou et al.A group of ent-Kaurane diterpenoids inhibit hedgehog signaling and induce cilia elongationPLoS ONE2015
    Henkels, Karen M. et al.S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid bindingFASEB Journal2015
    Lin, Wei Chi et al.Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideumMolecular Biology of the Cell2015
    Odermatt, Pascal D. et al.High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force MicroscopyNano Letters2015
    Lutz, Vanessa et al.SAR studies on hydropentalene derivatives - Important core units of biologically active tetramic acid macrolactams and ptychanolidesBioorganic and Medicinal Chemistry2014
    Chan, Eddie et al.The acetylenic tricyclic bis(cyano enone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actinCancer Prevention Research2014
    Speranza, Francis et al.The molecular basis of leukocyte adhesion involving phosphatidic acid and phospholipase DJournal of Biological Chemistry2014
    La Clair, James J. et al.In Situ natural product discovery via an artificial marine spongePLoS ONE2014
    Nievergelt, Adrian P. et al.High-frequency multimodal atomic force microscopyBeilstein Journal of Nanotechnology2014
    Yuan, Baiyin et al.A cardiomyocyte-specific Wdr1 knockout demonstrates essential functional roles for actin disassembly during myocardial growth and maintenance in miceAmerican Journal of Pathology2014
    Taniuchi, Keisuke et al.RUVBL1 directly binds actin filaments and induces formation of cell protrusions to promote pancreatic cancer cell invasionInternational Journal of Oncology2014
    Xiao, Xiang et al.c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution2013
    Jiwani, Shahanawaz et al.Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerizationBiochemical and Biophysical Research Communications2012
    Fan, Jianguo et al.A role for γs-crystallin in the organization of actin and fiber cell maturation in the mouse lensFEBS Journal2012
    Butler, Suzanne C. et al.Inhibitory effects of pectenotoxins from marine algae on the polymerization of various actin isoformsToxicology in Vitro2012
    Perisic, Ljubica et al.Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamicsKidney International2012
    Xavier, Charles Peter et al.Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migrationScientific Reports2012
    Trigili, Chiara et al.Mechanism of Action of the Cytotoxic Macrolides Amphidinolide X and JChemBioChem2011
    Tsai, Chih Chien et al.7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migrationBiochemical Pharmacology2011
    Peterson, Jon E. et al.Normal ranges of angiogenesis regulatory proteins in human plateletsAmerican Journal of Hematology2010
    Lai, Ning Sheng et al.Phostensin caps to the pointed end of actin filaments and modulates actin dynamicsBiochemical and Biophysical Research Communications2009
    Chellaiah, Meenakshi A. et al.Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorptionJournal of Biological Chemistry2007
    Nusco, Gilda A. et al.Modulation of calcium signalling by the actin-binding protein cofilinBiochemical and Biophysical Research Communications2006
    Takamiya, Rina et al.Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal changeAmerican journal of physiology. Cell physiology2005
    Chellaiah, Meenakshi A.Regulation of actin ring formation by Rho GTPases in osteoclastsJournal of Biological Chemistry2005
    Kumar, Narendra et al.Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villinThe Journal of biological chemistry2004
    Nielsen, Finn C. et al.Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domainsJournal of Cell Science2002
    Zhai, Liwei et al.Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeletonThe Journal of biological chemistry2001
    Fontao, Lionel et al.The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectinJournal of cell science2001
    Blader, Ira J. et al.GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitroMolecular Biology of the Cell1999

     

    Question 1: Can I use this polymerization kit to measure polymerization of non-muscle actin?

    Answer 1:  Pyrene-labeled non-muscle actin has been shown to be unstable under normal storage conditions.  To examine the polymerization of unlabeled non-muscle actin, please click here for a polymerization protocol that uses an excess of unlabeled non-muscle, cardiac or gizzard actin (Cat# APHL95, APHL99, AS99) + a small amount of pyrene-labeled skeletal muscle actin (Cat. # AP05).  The pyrene muscle actin will not polymerize efficiently on its own at the concentration used in this assay, so the reaction is dependent on unlabeled actin polymerization for F-actin formation.  In this way, the pyrene-labeled muscle actin is taken up and polymerized to serve as a reporter for polymerization of the unlabeled actin that is present at a much greater concentration. 

     

    Question 2:  Can cell lysates be used with this kit as the source of a test protein?

    Answer 2:  Yes, cell lysates can be used as the source of the test protein for examining actin polymerization enhancers and inhibitors.  However, Cytoskeleton does not recommend this as the purity and concentration of the protein will often be too low to interact with actin.  Also, the lysates will contain additional accessory proteins and multiple phosphatases and proteases that can interfere or alter the interactions between actin and test protein.  If lysates are to be used, we recommend the following:

    Although this kit is designed for use with pure proteins or compounds, some researchers have added extracts with good results. Generally researchers use over-expressed proteins and a wild-type control extract similarly over-expressed.  It is necessary to make a 10 mg/ml protein extract and then use 1/3rd volume of this to 2/3rd volume of pyrene-actin (Cat. # AP05).  In this way there is a high enough concentration of protein to make a difference.  The extraction buffer should be 20 mM Hepes pH 7.5, 20 mM NaCl, plus any co-factors for your protein, and a protease inhibitor cocktail (e.g., Cat# PIC02).  Rinse the cells with an ice cold buffer and lyse cells with a 25 g bent over syringe needle or other device.  The control cell line is very critical because the actin polymerization reaction is very sensitive to slight differences in protein concentration or salts. 

     

     

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

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