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. Actin polyclonal antibody (Cat. # AAN01)
  8. SDS sample buffer (5 x)
  9. DMSO
  10. Manual with detailed protocols and extensive troubleshooting guide 

Equipment needed

  1. Centrifuge capable of temperature controlled operation at 100,000 x g with volumes of 100 µl to 2 ml depending on the cell lysis volume
  2. SDS-PAGE minigel system and western blotting transfer apparatus

Example results
Changes in the amount of G-actin and F-actin were investigated in Swiss 3T3 cells treated with the actin polymerizing drug jasplakinolide, using the G-actin/F-actin in vivo assay kit.  In untreated Swiss 3T3 cells, 80% of actin is soluble G-actin, and is found within the supernatant fraction, 20% of actin is filamentous F-actin and is found in the pellet fraction. In Swiss 3T3 cells treated with jasplakinolide, 80% of actin is reorganized into F-actin and is found in the pellet fraction (Fig. 1).

Figure 1. Reorganization of actin in Swiss 3T3 cells after treatment with jasplakinolide. Swiss 3T3 cells were treated with jasplakinolide (Jaspl) or left untreated (Untr) and the G-actin (G) and F-actin (F) content was assayed using BK037. Treatment with jasplakinolide resulted in a potent accumulation of F-actin.

References

  1. Yassin, R., Shefcyk, J., White, J. R., Tao, W., Volpi, M., Molski, T. F. P., Naccache, P. H., Feinstein, M. B. and Sha’Afi, R. I. (1985). Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils. J. Cell Biol. 101, 182-188.
  2. White, J. R., Naccache, P. H., and Sha’Afi, R. I. (1983). Stimulation of chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils. Effects of calcium and cytochalasin B.  J. Biol. Chem. 258, 14041-14047.
  3. Hartwig, J. H. (1992). An ultrastructural approach to understanding the cytoskeleton. In The Cytoskeleton, A practical approach, Ed. K. L. Carraway and C. A. C. Carraway, Oxford University Press.
  4. Rao, K. M. K., Betschart, J. M. and Virji, M. A. (1985). Hormone induced actin polymerization in rat hepatoma cells and human leucocytes. Biochem. J. 230, 709-714.

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

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. 

 

 

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