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:
- Lysis and F-actin stabilization buffer
- ATP (Cat. # BSA04)
- Protease inhibitor cocktail (Cat. # PIC02)
- F-actin enhancing control solution
- F-actin depolymerization control solution
- Control G-actin Standard (Cat. # AKL99)
- Actin polyclonal antibody (Cat. # AAN01)
- SDS sample buffer (5 x)
- DMSO
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- 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
- 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).
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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. |
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References
- 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.
- 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.
- 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.
- 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
Product Description
MSDS
Datasheet
Cat #
G-Actin/F-actin In Vivo Assay Biochem Kit
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BK037
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