G-Actin : F-Actin In Vivo Assay 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. Anti-Actin MAb (clone 7A8.2.1) (Cat # AAN02-S)
8. SDS sample buffer (5 x)
9. DMSO
10. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

1. Temperature controlled centrifuge capable of reaching 100, 000 x g. Ideally accepts 100 ul sample volumes. The assay can be adapted for larger volumes, however, this may result in less assays per kit (see Section VI: Assay Protocol).
2. Small homogenizer suitable for low milliliter volumes or 25G needle and syringe.
3. SDS-PAGE and western blot apparatus and reagents, anti-mouse-HRP secondary Ab

Example results
Swiss 3T3 cells were grown to 50% confluency in DMEM / 10% FBS at 37C/5% CO2. Cells were untreated (lanes 1P and 1S) or treated with 0.1 uM of the actin polymerizing drug jasplakinolide for 30 minutes at 37C/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

1. Milligan R.A. et al. 1990. Molecular structure of F-actin and location of surface binding sites. Nature 348, 217-221.
2. Dos Remedios C.G. et al. 2003. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol. Rev. 83, 433-473.
3. Phillips D.R. et al. 1980. Identification of membrane proteins mediating the interaction of human platelets. J. Cell Biol. 86, 77-86.
4. 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.

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 infohoelzel.de