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. 
  

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.