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

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

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. 

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.


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). 


  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

Bojcevski, Jovana et al. “Influence of retinal NMDA receptor activity during autoimmune optic neuritis.” Journal of neurochemistry vol. 153,6 (2020): 693-709. doi:10.1111/jnc.14980

Chen, Y. et al.  "Coronin 2B regulates dendrite outgrowth by modulating actin dynamics." FEBS Lett, 594: 2975-2987. (2020). https://doi.org/10.1002/1873-3468.13886

Yang, N. et al. Effects of dexamethasone on remodeling of the hippocampal synaptic filamentous actin cytoskeleton in a model of pilocarpine-induced status epilepticus. Int. J. Med. Sci. 17, 1683–1691 (2020).

Gupta, R., Kumar, G., Jain, B. P., Chandra, S. & Goswami, S. K. Ectopic expression of 35 kDa and knocking down of 78 kDa SG2NAs induce cytoskeletal reorganization, alter membrane sialylation, and modulate the markers of EMT. Mol. Cell. Biochem. (2020) doi:10.1007/s11010-020-03932-2.

Musah, A. S. et al. Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination. J. Neurosci. 40, 2993 LP – 3007 (2020).

Shi, Zengdun et al. “Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesis.” American journal of physiology. Gastrointestinal and liver physiology vol. 318,3 (2020): G504-G517. doi:10.1152/ajpgi.00302.2019

Yang, Nuo et al. “Effects of Dexamethasone on Remodeling of the Hippocampal Synaptic Filamentous Actin Cytoskeleton in a Model of Pilocarpine-induced Status Epilepticus.” International journal of medical sciences vol. 17,12 1683-1691. 2 Jul. 2020, doi:10.7150/ijms.44927

Bojcevski J, Stojic A, Hoffmann DB, Williams SK, Müller A, Diem R, Fairless R. Influence of retinal NMDA receptor activity during autoimmune optic neuritis. J Neurochem. 2020 Jun;153(6):693-709. doi: 10.1111/jnc.14980. Epub 2020 Mar 3. PMID: 32031240.

Kommaddi, Reddy Peera et al. “Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 38,5 (2018): 1085-1099. doi:10.1523/JNEUROSCI.2127-17.2017

Kommaddi RP et al. Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease. J Neurosci. 2018 Jan 31;38(5):1085-1099. doi: 10.1523/JNEUROSCI.2127-17.2017. Epub 2017 Dec 15. PMID: 29246925; PMCID: PMC5792472.

Asahara et al., 2013. Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin. Diabetologia. doi: 10.1007/s00125-013-2849-5.

Ni et al., 2013. The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosis. Free Radical Biology and Medicine. doi: http://dx.doi.org/10.1016/j.freeradbiomed.2013.03.012.

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Shuang et al., 2013. Destrin deletion enhances the bone loss in hindlimb suspended mice. E. J. Appl. Physiol. 113, 403-410.

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Fan et al., 2012. A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens. FEBS. J. 279, 2892-2904.

Liu et al., 2012. TLR2 Is a Primary Receptor for Alzheimer's Amyloid β Peptide To Trigger Neuroinflammatory Activation. J. Immunol. 188, 1098-1107.

Chand et al., 2012. C-terminal region of teneurin-1 co-localizes with dystroglycan and modulates cytoskeletal organization through an extracellular signal-regulated kinase-dependent stathmin- and filamin A-mediated mechanism in hippocampal cells. Neuroscience. 219, 255-270.

Rapier et al., 2010. The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell Int. 10, 24.

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Zhang et al., 2005. Activation of the Arp2/3 complex by N-WASP is required for actin polymerization and contraction in smooth muscle. Am. J. Physiol. 288, C1145-C1160.

Chen et al., 2004. Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J. Biol. Chem. 279, 39705-39709.

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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