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
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
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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
Cytoskeleton公司成立于1993年，专注于生物化学和细胞过程研究中的纯化蛋白和便捷试剂盒开发与生产。公司提供药物筛选、信号转导、细胞骨架研究相关的系列试剂盒和产品，尤其以细胞骨架相关研究见长，既能满足于样品较少的科学研究，也可以用于小规模筛选研究和高通量大规模筛选研究。此外，公司还提供微管蛋白，肌动蛋白，小G蛋白，GAPs，GEFs等现有产品的药物筛选服务。
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