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

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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
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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
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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
Liu, Zhenan et al.Control of Podocyte and Glomerular Capillary Wall Structure and Elasticity by WNK1 KinaseFrontiers in Cell and Developmental Biology2021ISSN 2296-634X
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
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
Bojcevski, Jovana et al.Influence of retinal NMDA receptor activity during autoimmune optic neuritisJournal of Neurochemistry2020ISSN 1471-4159
Sankaran, Jeyantt S. et al.Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cellsStem Cells2020ISSN 1549-4918
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
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
Yuki, Kyoko E. et al.CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infectionNature Microbiology2019ISSN 2058-5276
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
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
Chen, Wei et al.The phenotype of peritoneal mouse macrophages depends on the mitochondria and ATP/ADP homeostasisCellular Immunology2018ISSN 1090-2163
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
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
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Allison, Patrick et al.BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinaseCell Adhesion and Migration2016ISSN 1933-6926
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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
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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