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.
IntroductionThe 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 contentsThe 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)
- Anti-Actin MAb (clone 7A8.2.1) (Cat # AAN02-S)
- SDS sample buffer (5 x)
- DMSO
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- 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).
- Small homogenizer suitable for low milliliter volumes or 25G needle and syringe.
- SDS-PAGE and western blot apparatus and reagents, anti-mouse-HRP secondary Ab
Example resultsSwiss 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).
References
- Milligan R.A. et al. 1990. Molecular structure of F-actin and location of surface binding sites. Nature 348, 217-221.
- Dos Remedios C.G. et al. 2003. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol. Rev. 83, 433-473.
- Phillips D.R. et al. 1980. Identification of membrane proteins mediating the interaction of human platelets. J. Cell Biol. 86, 77-86.
- 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
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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
