Product Uses Include
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.
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:
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).
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.
Ramachandran et al., 2013. JunB mediates basal- and TGFb1-induced smooth muscle cell contractility. PLoS ONE. 8(1): e53430.
Shuang et al., 2013. Destrin deletion enhances the bone loss in hindlimb suspended mice. E. J. Appl. Physiol. 113, 403-410.
Malenda et al., 2012. Statins Impair Glucose Uptake in Tumor Cells. Neoplasia. 14, 311–323.
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.
Meeks et al., 2005. Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery. Am. J. Physiol. 288, C633-C639.
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.
Tang and Gunst, 2004. The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728.
Searles et al., 2004. Actin cytoskeleton organization and poststranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circ. Res. 95, 488-495.
Tu et al., 2003. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell. 113, 37-47.
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 firstname.lastname@example.org