G-Actin/F-actin In Vivo Assay Biochem Kit

G-Actin/F-actin In Vivo Assay Biochem Kit
$0.00

Product Uses Include

  • To study the effects of pharmaceutical compounds on the ratio of G-actin to F-actin.
  • To study the effects of mutated cell lines versus their parent cell line for the change in ratio of G-actin to F-actin.
  • To study the effects of physical alterations of environment on the ratio of G-actin to F-actin. 

Introduction
The most reproducible and accurate method of determining the amount of filamentous actin (F-actin) content versus free globular-actin (G-actin) content in a cell population is to use Western blot quantitation of F-actin and G-actin cellular fractions (1-4).  The general approach is to homogenize cells in F-actin stabilization buffer, followed by centrifugation to separate the F-actin from G-actin pool. The fractions are then separated by SDS-PAGE and actin is quantitated by Western blot. The final result gives the most accurate method of determining the ratio of F-actin incorporated into the cytoskeleton versus the G-actin found in the cytosol. This kit contains all the reagents needed to perform this assay.

Kit contents
The kit contains sufficient materials for 30-100 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:

  1. Lysis and F-actin stabilization buffer
  2. ATP (Cat. # BSA04)
  3. Protease inhibitor cocktail (Cat. # PIC02)
  4. F-actin enhancing control solution
  5. F-actin depolymerization control solution
  6. Control G-actin Standard (Cat. # AKL99)
  7. Anti-Actin MAb (clone 7A8.2.1) (Cat # AAN02-S)
  8. SDS sample buffer (5 x)
  9. DMSO
  10. Manual with detailed protocols and extensive troubleshooting guide 

Equipment needed

  1. Temperature controlled centrifuge capable of reaching 100,000 x g. Ideally accepts 100 µl sample volumes. The assay can be adapted for larger volumes, however, this may result in less assays per kit (see Section VI: Assay Protocol).
  2. Small homogenizer suitable for low milliliter volumes or 25G needle and syringe.
  3. SDS-PAGE and western blot apparatus and reagents, anti-mouse-HRP secondary Ab

Example results
Swiss 3T3 cells were grown to 50% confluency in DMEM / 10% FBS at 37°C/5% CO2.  Cells were untreated  (lanes 1P and 1S) or treated with 0.1 µM of the actin polymerizing drug jasplakinolide for 30 minutes at 37°C/5% CO2 (lanes 2P and 2S).  Cells were lysed and processed into supernatant (S) and pellet (P) fractions and ana-lysed by western blot quantitation of actin protein according to the G-actin/F-actin In Vivo Assay Kit instructions.

BK037_Figure_1

Panel 1: In untreated Swiss 3T3 cells, 45% of actin is soluble G-actin (1S) and 55% is insoluble F-actin (1P). This agrees with published data (3).Panel 2: In Swiss 3T3 cells treated with the actin polymerizing drug jasplakinolide, only 5% of actin remains in the soluble G-actin fraction (2S) while 95% is found in the insoluble F-actin pellet fraction (2P). Lanes 50, 20 and 10 represents 50ng, 20ng and 10 ng of G-actin standard. M represents molecular weight markers (molecular weights are shown to the right of the blot). 

References

  1. Milligan R.A. et al. 1990. Molecular structure of F-actin and location of surface binding sites. Nature 348, 217-221.
  2. Dos Remedios C.G. et al. 2003. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol. Rev. 83, 433-473.
  3. Phillips D.R. et al. 1980. Identification of membrane proteins mediating the interaction of human platelets. J. Cell Biol. 86, 77-86.
  4. Kim H.R. et al. 2008. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent. Am. J. Physiol. Cell Physiol. 295, C768-C778.

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
Kamnev, Anton et al.Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cellsCell reports2024ISSN 2211--1247
Yin, Xiufeng et al.CFL1 is Implicated in Chronic Myeloid Leukemia Response during Imatinib TherapyJournal of Cancer2024ISSN 1837-9664
Medina-Dols, Aina et al.Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axisCell Death Discovery 2024 10:12024ISSN 2058--7716
Li, Yuan Bao et al.Fimbrin associated with Pmk1 to regulate the actin assembly during Magnaporthe oryzae hyphal growth and infectionStress Biology2024ISSN 2731-0450
Wang, Lingling et al.Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actinReproductive Biology and Endocrinology 2024 22:12024ISSN 1477--7827
Yu, Jinhan et al.Inhibiting F-Actin Polymerization Impairs the Internalization of Moraxella catarrhalisMicroorganisms 2024, Vol. 12, Page 2912024ISSN 2076--2607
Kinnart, Inge et al.Elevated α-synuclein levels inhibit mitophagic fluxnpj Parkinson's Disease 2024 10:12024ISSN 2373--8057
Kneppers, Anita et al.AMPKα2 is a skeletal muscle stem cell intrinsic regulator of myonuclear accretioniScience2023ISSN 2589--0042
Chen, Wei Ju et al.Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis PathwayAdvanced Science2023ISSN 2198--3844
Nguyen, Abraham et al.TTBK2 controls cilium stability by regulating distinct modules of centrosomal proteinsMolecular Biology of the Cell2023ISSN 1939-4586
Hijazi, Nour et al.Paxillin regulates liver fibrosis via actin polymerization and ERK activation in hepatic stellate cellsJournal of Cell Science2023ISSN 1477-9137
Li, Zixin et al.Self-promoted electroactive biomimetic mineralized scaffolds for bacteria-infected bone regenerationNature Communications 2023 14:12023ISSN 2041--1723
Makarova, Nadezda et al.Mechanical Way To Study Molecular Structure of Pericellular LayerACS Applied Materials and Interfaces2023ISSN 1944-8252
Guan, Linbo et al.Lymphangiogenic responses of lymphatic endothelial cells to steady direct-current electric fieldsCell Adhesion & Migration2023ISSN 1933-6926
Polino, Alexander J. et al.Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeletonJournal of Biological Chemistry2023ISSN 1083-351X
Rakotomamonjy, Jennifer et al.PCDH12 loss results in premature neuronal differentiation and impeded migration in a cortical organoid modelCell reports2023ISSN 2211--1247
Du, William W. et al.Nuclear Actin Polymerization Regulates Cell Epithelial-Mesenchymal TransitionAdvanced Science2023ISSN 2198--3844
Levy-Myers, Reuben et al.An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic functionScience Advances2023ISSN 2375-2548
St. Paul, Amanda et al.FXR1 regulates vascular smooth muscle cell cytoskeleton, VSMC contractility, and blood pressure by multiple mechanismsCell reports2023ISSN 2211-1247
Wu, Qiaoyun et al.Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNLBiological Research2023ISSN 0717-6287
Wang, Caiqin et al.Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancerJournal of Translational Medicine 2023 21:12023ISSN 1479--5876
Correa, Francisco et al.Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during PostconditioningCells2023ISSN 2073-4409
Romero, Luis O. et al.Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman SyndromeNature Communications 2023 14:12023ISSN 2041--1723
Bogucka-Janczi, Katarzyna et al.ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.eLife2023ISSN 2050--084X
Li, Linxi et al.PCP Protein Inversin Regulates Testis Function Through Changes in Cytoskeletal Organization of Actin and MicrotubulesEndocrinology2022ISSN 0013--7227
Si, Hongjiang et al.A Highlights from MBoC Selection: Noncanonical Wnt/Ror2 signaling regulates cell–matrix adhesion to prompt directional tumor cell invasion in breast cancerMolecular Biology of the Cell2022ISSN 1939-4586
Flores-Muñoz, Carolina et al.The Long-Term Pannexin 1 Ablation Produces Structural and Functional Modifications in Hippocampal NeuronsCells2022ISSN 2073-4409
Lohraseb, Iman et al.Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cellsNature Communications 2022 13:12022ISSN 2041--1723
Fang, Shi et al.RhoBTB1 reverses established arterial stiffness in angiotensin II–induced hypertension by promoting actin depolymerizationJCI Insight2022ISSN 0021--9738
He, Zheng Quan et al.Pharmacological regulation of tissue fibrosis by targeting the mechanical contraction of myofibroblastsFundamental Research2022ISSN 2667-3258
Garcia, Alix et al.miR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIaThrombosis and haemostasis2021ISSN 2567--689X
Misra, Suniti et al.Periostin/Filamin-A: A Candidate Central Regulatory Axis for Valve Fibrogenesis and Matrix CompactionFrontiers in Cell and Developmental Biology2021ISSN 2296-634X
Saal, Kim Ann et al.Rho-kinase inhibition by fasudil modulates pre-synaptic vesicle dynamicsJournal of Neurochemistry2021ISSN 1471--4159
Balbi, Carolina et al.An exosomal-carried short periostin isoform induces cardiomyocyte proliferationTheranostics2021ISSN 1838--7640
Zhang, Chong et al.Fibrinogen/AKT/Microfilament Axis Promotes Colitis by Enhancing Vascular PermeabilityCellular and molecular gastroenterology and hepatology2021ISSN 2352--345X
Gupta, Richa et al.Ectopic expression of 35 kDa and knocking down of 78 kDa SG2NAs induce cytoskeletal reorganization, alter membrane sialylation, and modulate the markers of EMTMolecular and cellular biochemistry2021ISSN 1573--4919
Kim, Ji Eun et al.PLPP/CIN-mediated NF2-serine 10 dephosphorylation regulates F-actin stability and Mdm2 degradation in an activity-dependent mannerCell Death and Disease2021ISSN 2041-4889
Landry, Natalie M. et al.SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activationBasic Research in Cardiology2021ISSN 1435-1803
Morandell, Jasmin et al.Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain developmentNature Communications2021ISSN 2041-1723
Guerrero-Valero, Marta et al.Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathyProceedings of the National Academy of Sciences of the United States of America2021ISSN 1091-6490
Yang, Nuo et al.Effects of dexamethasone on remodeling of the hippocampal synaptic filamentous actin cytoskeleton in a model of pilocarpine-induced status epilepticusInternational Journal of Medical Sciences2020ISSN 1449-1907
Lu, Yi Ju et al.Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunityNature Communications2020ISSN 2041-1723
Chen, Yuewen et al.Coronin 2B regulates dendrite outgrowth by modulating actin dynamicsFEBS Letters2020ISSN 1873-3468
Huang, Rui et al.NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesisJournal of Cell Biology2020ISSN 1540-8140
Shi, Zengdun et al.Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesisAmerican Journal of Physiology - Gastrointestinal and Liver Physiology2020ISSN 1522-1547
Luo, Xi et al.Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axisCell and Bioscience2020ISSN 2045-3701
Kitchen, Gareth B. et al.The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumoniaProceedings of the National Academy of Sciences of the United States of America2020ISSN 1091-6490
Angelini, Aude et al.Cardioprotective effects of α-cardiac actin on oxidative stress in a dilated cardiomyopathy mouse modelFASEB Journal2020ISSN 1530-6860
Hogrebe, Nathaniel J. et al.Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cellsNature Biotechnology2020ISSN 1546-1696
Musah, Aminat S. et al.Mechanistic target of rapamycin regulates the oligodendrocyte cytoskeleton during myelinationJournal of Neuroscience2020ISSN 1529-2401
Wenceslau, Camilla F. et al.Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerizationPharmacological Research2019ISSN 1096-1186
Miller, Karl N. et al.PGC-1a integrates a metabolism and growth network linked to caloric restrictionAging Cell2019ISSN 1474-9726
Hu, Fang et al.Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuronFASEB Journal2019ISSN 1530-6860
Yu, Minhao et al.Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathwayEBioMedicine2019ISSN 2352-3964
Rockey, Don C. et al.Smooth Muscle α-Actin Deficiency Leads to Decreased Liver Fibrosis via Impaired Cytoskeletal Signaling in Hepatic Stellate CellsAmerican Journal of Pathology2019ISSN 1525-2191
Torrini, Consuelo et al.Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte ProliferationCell Reports2019ISSN 2211-1247
Childers, Rachel C. et al.Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failureAmerican Journal of Physiology - Heart and Circulatory Physiology2019ISSN 1522-1539
Burger, Joyce et al.Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFβ signalingCellular Signalling2019ISSN 1873-3913
Wen, Qing et al.Myosin VIIa supports spermatid/organelle transport and cell adhesion during spermatogenesis in the rat testisEndocrinology2019ISSN 1945-7170
Zhao, Fei et al.Exendin-4 promotes actin cytoskeleton rearrangement and protects cells from Nogo-A-Δ20 mediated spreading inhibition and growth cone collapse by down-regulating RhoA expression and activation via the PI3K pathwayBiomedicine and Pharmacotherapy2019ISSN 1950-6007
Kommaddi, Reddy Peera et al.Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in alzheimer’s diseaseJournal of Neuroscience2018ISSN 1529-2401
Wen, Qing et al.Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testisCell Death and Disease2018ISSN 2041-4889
Bardai, Farah H. et al.A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivoJournal of Neuroscience2018ISSN 1529-2401
Cahill, Michael E. et al.Withdrawal from repeated morphine administration augments expression of the RhoA network in the nucleus accumbens to control synaptic structureJournal of Neurochemistry2018ISSN 1471-4159
Zhang, Fanrong et al.GAP43, a novel metastasis promoter in non-small cell lung cancer 11 Medical and Health Sciences 1112 Oncology and CarcinogenesisJournal of Translational Medicine2018ISSN 1479-5876
Mauriac, Stephanie A. et al.Defective Gpsm2/Gαi3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndromeNature Communications2017ISSN 2041-1723
Tavares, Sandra et al.Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cellsNature Communications2017ISSN 2041-1723
Pérez-Núñez, Ramón et al.Overexpressed Down Syndrome Cell Adhesion Molecule (DSCAM) Deregulates P21-Activated Kinase (PAK) Activity in an In Vitro Neuronal Model of Down Syndrome: Consequences on Cell Process Formation and ExtensionNeurotoxicity Research2016ISSN 1476-3524
Allison, Patrick et al.BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinaseCell Adhesion and Migration2016ISSN 1933-6926
Zhang, Shu Xin et al.Actin Aggregations Mark the Sites of Neurite InitiationNeuroscience Bulletin2016ISSN 1995-8218
Freeman, Spencer A. et al.Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptorNature Communications2015ISSN 2041-1723
Peykov, S. et al.Identification and functional characterization of rare SHANK2 variants in schizophreniaMolecular Psychiatry2015ISSN 1476-5578
Allison, Patrick et al.Type III TGFβ receptor and Src direct hyaluronan-mediated invasive cell motilityCellular Signalling2015ISSN 1873-3913
Caution, Kyle et al.Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearanceScientific Reports2015ISSN 2045-2322
Wang, J. et al.Erratum: The small GTPase RhoA, but not Rac1, is essential for conditioned aversive memory formation through regulation of actin rearrangements in rat dorsal hippocampus (Acta Pharmacol Sin (2013) 34 (811-8) Doi: 10.1038/aps.2013.3 )Acta Pharmacologica Sinica2014ISSN 1745-7254
Kwok, Willson et al.Rho-kinase activation contributes to lps-induced impairment of endothelial nitric oxide synthase activation by endothelin-1 in cultured hepatic sinusoidal endothelial cellsShock2014ISSN 1540-0514
Asahara, S. et al.Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actinDiabetologia2013PMID 23412604
Ramachandran, Aruna et al.JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell ContractilityPLOS ONE2013ISSN 1932--6203
Shuang, Feng et al.Destrin deletion enhances the bone loss in hindlimb suspended miceEuropean journal of applied physiology2013ISSN 1439--6327
Stehn, Justine R. et al.A novel class of anticancer compounds targets the actin cytoskeleton in tumor cellsCancer Research2013ISSN 0008-5472
Fan, Jianguo et al.A role for γs-crystallin in the organization of actin and fiber cell maturation in the mouse lensFEBS Journal2012ISSN 1742-464X
Malenda, Agata et al.Statins Impair Glucose Uptake in Tumor CellsNeoplasia2012ISSN 1476--5586
Liu, Shirong et al.TLR2 is a primary receptor for Alzheimer's amyloid β peptide to trigger neuroinflammatory activationJournal of immunology (Baltimore, Md. : 1950)2012ISSN 1550--6606
Varol, Başak et al.The cytotoxic effect of diphtheria toxin on the actin cytoskeletonCellular and Molecular Biology Letters2012ISSN 1689-1392
Shen, Dongxiao et al.Aortic aneurysm generation in mice with targeted deletion of integrin-linked kinase in vascular smooth muscle cellsCirculation Research2011ISSN 0009-7330
Rapier, Rebecca et al.The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cellsCancer Cell International2010ISSN 1475-2867
Romero, Ana M. et al.Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary cultureToxicological Sciences2010ISSN 1096-6080
Tejani, Ankit D. et al.Force augmentation and stimulated actin polymerization in swine carotid arteryAmerican Journal of Physiology - Cell Physiology2010ISSN 0363-6143
Du, Hui et al.Phenylephrine induces elevated RhoA activation and smooth muscle α-actin expression in Pkd2+/-vascular smooth muscle cellsHypertension Research2010ISSN 0916-9636
McArthur, Simon et al.Annexin A1 regulates hormone exocytosis through a mechanism involving actin reorganizationFASEB Journal2009ISSN 1530-6860
Fu, Xiao Dong et al.Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeletonPLoS ONE2008ISSN 1932-6203
Bergstrom, R. A. et al.Agrin induced morphological and structural changes in growth cones of cultured hippocampal neuronsNeuroscience2007ISSN 0306-4522
Meeks, Melissa K. et al.Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid arteryAmerican journal of physiology. Cell physiology2005ISSN 0363--6143
Zhang, Wenwu et al.Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscleAmerican journal of physiology. Cell physiology2005ISSN 0363--6143
Sebastian, Becky M. et al.Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytesAmerican Journal of Physiology - Endocrinology and Metabolism2005ISSN 0193-1849
Searles, Charles D. et al.Actin cytoskeleton organization and posttranscriptional regulation of endothelial nitric oxide synthase during cell growthCirculation research2004ISSN 1524--4571
Tang, Dale D. et al.The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscleJournal of Biological Chemistry2004ISSN 0021-9258
Chen, Guoli et al.Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulinJournal of Biological Chemistry2004ISSN 0021-9258
Tang, Dale D. et al.Role of Crk-Associated Substrate in the Regulation of Vascular Smooth Muscle ContractionHypertension2003ISSN 1524-4563
Tu, Yizeng et al.Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulationCell2003ISSN 0092-8674

Question 1:  At which step can the assay be stopped?

Answer 1:  The assay cannot be stopped until after the 100,000 x g spin for 1 hour at 37°C.  After this high speed centrifugation, the supernatant (G-actin) can be mixed with SDS loading buffer and frozen for later use.  The pellet (F-actin) should be resuspended with a depolymerizing agent and water and then mixed with SDS loading buffer and frozen for later use.  Upon freezing, F-actin depolymerizes, so it is necessary to separate the F-actin from the G-actin before freezing samples to isolate samples for an accurate measurement of F-actin and G-actin ratios.

 

Question 2:  How sensitive is this assay?

Answer 2:  The assay can detect as small as a 15% shift in G-actin to F-actin ratio.  Each condition should be performed in duplicate and repeated several times as assay reproducibility can vary by 10-20% between experiments.

 

Question 3: Will the kit work at slower centrifugation speeds such as 16,000 x g?

Answer 3: Unfortunately, no. In our testing centrifugation speeds slower than 100,000 x g, including up to 26,000 x g, failed to efficiently pellet F-actin. 

 

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