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
Zhang, Yan et al.Different changes in pre- and postsynaptic components in the hippocampal CA1 subfield after transient global cerebral ischemiaBrain Structure and Function2022ISSN 1863-2661
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
He, Zheng Quan et al.Pharmacological regulation of tissue fibrosis by targeting the mechanical contraction of myofibroblastsFundamental Research2022ISSN 2667-3258
Fang, Shi et al.RhoBTB1 reverses established arterial stiffness in angiotensin II–induced hypertension by promoting actin depolymerizationJCI Insight2022ISSN 0021--9738
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
Landry, Natalie M. et al.SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activationBasic Research in Cardiology2021ISSN 1435-1803
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
Zhang, Chong et al.Fibrinogen/AKT/Microfilament Axis Promotes Colitis by Enhancing Vascular PermeabilityCellular and molecular gastroenterology and hepatology2021ISSN 2352--345X
Balbi, Carolina et al.An exosomal-carried short periostin isoform induces cardiomyocyte proliferationTheranostics2021ISSN 1838--7640
Saal, Kim Ann et al.Rho-kinase inhibition by fasudil modulates pre-synaptic vesicle dynamicsJournal of Neurochemistry2021ISSN 1471--4159
Liu, Zhenan et al.Control of Podocyte and Glomerular Capillary Wall Structure and Elasticity by WNK1 KinaseFrontiers in Cell and Developmental Biology2021ISSN 2296-634X
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
Lee, Minseon et al.Hypergravity-induced changes in actin response of breast cancer cells to natural killer cellsScientific Reports2021ISSN 2045-2322
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
Morandell, Jasmin et al.Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain developmentNature Communications2021ISSN 2041-1723
Garcia, Alix et al.miR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIaThrombosis and haemostasis2021ISSN 2567--689X
Bojcevski, Jovana et al.Influence of retinal NMDA receptor activity during autoimmune optic neuritisJournal of Neurochemistry2020ISSN 1471-4159
Li, Huitao et al.NC1-Peptide from collagen α3 (IV) chains in the basement membrane of testes regulates spermatogenesis via p-FAK-Y407Endocrinology (United States)2020ISSN 1945-7170
Jansson, Thomas et al.Down-regulation of placental Cdc42 and Rac1 links mTORC2 inhibition to decreased trophoblast amino acid transport in human intrauterine growth restrictionClinical Science2020ISSN 1470-8736
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
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
Musah, Aminat S. et al.Mechanistic target of rapamycin regulates the oligodendrocyte cytoskeleton during myelinationJournal of Neuroscience2020ISSN 1529-2401
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
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
Chen, Yuewen et al.Coronin 2B regulates dendrite outgrowth by modulating actin dynamicsFEBS Letters2020ISSN 1873-3468
Lu, Yi Ju et al.Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunityNature Communications2020ISSN 2041-1723
Sankaran, Jeyantt S. et al.Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cellsStem Cells2020ISSN 1549-4918
Wang, Yan et al.CD44 inhibits α-SMA gene expression via a novel G-actin/MRTF-mediated pathway that intersects with TGFβR/ p38MAPK signaling in murine skin fibroblastsJournal of Biological Chemistry2019ISSN 1083-351X
Miller, Karl N. et al.PGC-1a integrates a metabolism and growth network linked to caloric restrictionAging Cell2019ISSN 1474-9726
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
Wen, Qing et al.Myosin VIIa supports spermatid/organelle transport and cell adhesion during spermatogenesis in the rat testisEndocrinology2019ISSN 1945-7170
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
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
Burger, Joyce et al.Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFβ signalingCellular Signalling2019ISSN 1873-3913
Yuki, Kyoko E. et al.CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infectionNature Microbiology2019ISSN 2058-5276
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
Torrini, Consuelo et al.Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte ProliferationCell Reports2019ISSN 2211-1247
Yu, Minhao et al.Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathwayEBioMedicine2019ISSN 2352-3964
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
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
Chen, Wei et al.The phenotype of peritoneal mouse macrophages depends on the mitochondria and ATP/ADP homeostasisCellular Immunology2018ISSN 1090-2163
Chiu, Amy Pei Ling et al.Dual effects of hyperglycemia on endothelial cells and cardiomyocytes to enhance coronary LPL activityAmerican Journal of Physiology - Heart and Circulatory Physiology2018ISSN 1522-1539
Bardai, Farah H. et al.A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivoJournal 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
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
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
Tavares, Sandra et al.Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cellsNature Communications2017ISSN 2041-1723
Mauriac, Stephanie A. et al.Defective Gpsm2/Gαi3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndromeNature Communications2017ISSN 2041-1723
Halim, Danny et al.Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and miceProceedings of the National Academy of Sciences of the United States of America2017ISSN 1091-6490
González-Jamett, Arlek M. et al.Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cellsScientific Reports2017ISSN 2045-2322
Laguesse, Sophie et al.Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and RewardNeuron2017ISSN 1097-4199
Zhou, Ning et al.Rho Kinase Regulates Aortic Vascular Smooth Muscle Cell Stiffness Via Actin/SRF/Myocardin in HypertensionCellular Physiology and Biochemistry2017ISSN 1421-9778
Liu, Zhenan et al.Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblastsProceedings of the National Academy of Sciences of the United States of America2017ISSN 1091-6490
Oppliger, Joel et al.Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of MacropinocytosisJournal of Virology2016ISSN 0022--538X
Hien, Tran Thi et al.Elevated glucose levels promote contractile and cytoskeletal gene expression in vascular smooth muscle via rho/protein kinase C and actin polymerizationJournal of Biological Chemistry2016ISSN 1083-351X
Zhang, Shu Xin et al.Actin Aggregations Mark the Sites of Neurite InitiationNeuroscience Bulletin2016ISSN 1995-8218
Allison, Patrick et al.BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinaseCell Adhesion and Migration2016ISSN 1933-6926
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
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
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
Han, Yu et al.Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signalingJournal of Neurochemistry2015ISSN 1471-4159
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
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
Lockett, Stephen et al.Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticalsCytometry Part A2014ISSN 1552-4930
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
Huang, Xun et al.JG6, a novel marine-derived oligosaccharide, suppresses breast cancer metastasis via binding to cofilinOncotarget2014ISSN 1949-2553
Ni, Jixiang et al.The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosisFree Radical Biology and Medicine2013ISSN 0891-5849
Asahara, S. et al.Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actinDiabetologia2013PMID 23412604
Stehn, Justine R. et al.A novel class of anticancer compounds targets the actin cytoskeleton in tumor cellsCancer Research2013ISSN 0008-5472
Shuang, Feng et al.Destrin deletion enhances the bone loss in hindlimb suspended miceEuropean journal of applied physiology2013ISSN 1439--6327
Ramachandran, Aruna et al.JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell ContractilityPLOS ONE2013ISSN 1932--6203
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
Durand, C. M. et al.SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanismMolecular Psychiatry2012ISSN 1359-4184
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
Agrawal, Pankaj B. et al.Normal myofibrillar development followed by progressive sarcomeric disruption with actin accumulations in a mouse Cfl2 knockout demonstrates requirement of cofilin-2 for muscle maintenanceHuman Molecular Genetics2012ISSN 0964-6906
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
Tejani, Ankit D. et al.Force augmentation and stimulated actin polymerization in swine carotid arteryAmerican Journal of Physiology - Cell Physiology2010ISSN 0363-6143
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
Du, Hui et al.Phenylephrine induces elevated RhoA activation and smooth muscle α-actin expression in Pkd2+/-vascular smooth muscle cellsHypertension Research2010ISSN 0916-9636
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
Ambrosino, Concetta et al.Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor α in human breast cancer cell nucleiMolecular and Cellular Proteomics2010ISSN 1535-9476
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
McCarthy, Alicia M. et al.Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transportAmerican Journal of Physiology - Cell Physiology2006ISSN 0363-6143
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
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
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
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.The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscleJournal of Biological Chemistry2004ISSN 0021-9258
Searles, Charles D. et al.Actin cytoskeleton organization and posttranscriptional regulation of endothelial nitric oxide synthase during cell growthCirculation research2004ISSN 1524--4571
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
Tang, Dale D. et al.Role of Crk-Associated Substrate in the Regulation of Vascular Smooth Muscle ContractionHypertension2003ISSN 1524-4563
Cenni, V. et al.Targeting of the Akt/PKB kinase to the actin skeletonCellular and Molecular Life Sciences2003ISSN 1420-682X

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