G-Actin : F-Actin Assay Kit

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.  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 nr BSA04)
3. Protease inhibitor cocktail
4. F-actin enhancing control solution
5. F-actin depolymerization control solution
6. Control G-actin Standard (Cat nr AKL99)
7. Actin polyclonal antibody (Cat nr AAN01)
8. SDS sample buffer (5 x)
9. DMSO
10. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

1. 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
2. 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 in the Gallery, Related Downloads section).

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.