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
