G-Actin : F-Actin In Vivo Assay Kit

AT

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.

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.

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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).