To assay actin, 107 cells per condition were collected for fractionation of nuclear proteins and assay using “G-actin/F-actin Assay Kit” (Cytoskeleton, Inc., Denver CO). As per instructions, 100 ml LAS2 buffer was added to nuclear pellets for homogenization through aspiration in a 25-G syringe; lysates were incubated at 37°C for 10 minutes then centrifuged at 350g for 5 minutes at RT. Supernatant, containing G-actin, was then collected after 100,000g at 37°C for 1 hour. F-actin depolymerization buffer of 100 ml is added to the pellet to extract F-actin.
G actin f actin assay kit
Manufactured by Cytoskeleton
The G-actin/F-actin Assay Kit is a laboratory product designed to quantify the levels of monomeric (G-actin) and filamentous (F-actin) forms of the actin protein in cellular samples. It provides a reliable and efficient method to measure the dynamics of the actin cytoskeleton, which is essential for various cellular processes.
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8 protocols using g actin f actin assay kit
1
Quantifying Cellular Actin Dynamics
2
Quantifying Actin Dynamics
3
Actin Cytoskeleton Reorganization Assay
4
Quantifying Cellular Actin Dynamics
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To assay actin, 107 cells per condition were collected for fractionation of nuclear proteins and assay using “G-actin/F-actin Assay Kit” (Cytoskeleton, Inc., Denver CO). As per instructions, 100 μl LAS2 buffer was added to nuclear pellets for homogenization through aspiration in a 25-G syringe; lysates were incubated at 37 °C for 10 min then centrifuged at 350 g for 5 min at RT. Supernatant, containing G-actin, was then collected after 100,000 g at 37 °C for 1 h. F-actin depolymerization buffer of 100 μl is added to the pellet to extract F-actin.
5
Quantifying Cellular F-actin and G-actin
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The concentration of F-actin and G-actin in cells was obtained using an F-actin/G-actin assay kit (catalog number: BK 037, Cytoskeleton, Denver, CO). Briefly, cells were rinsed with phosphate-buffered saline at 25 °C and scraped and homogenized in a lysis and F-actin stabilization buffer (LAS1). F-actin was then separated from G-actin by centrifugation at 100,000 × g for 60 min at 37 °C. The F-actin-containing pellet was resuspended in ddH2O containing 2 μm cytochalasin D at a volume equivalent to the G-actin-containing supernatant volume. The resuspended F-actin pellet was kept on ice for 60 min with mixing by pipette every 15 min to dissociate F-actin. After dissociation, dissociated F-actin was centrifuged at 14,000 g for 10 min at 4 °C. The F-actin and G-actin preparations were then assayed for protein.
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Quantifying F-actin and G-actin in Gastric Smooth Muscle Cells
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Quantification of Cellular F-actin and G-actin
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F-actin and G-actin fractions were obtained using an F-actin/G-actin assay kit (BK 037, Cytoskeleton). Cells were scraped in LAS2 buffer containing detergents to disrupt the cell membrane before they were gently homogenized to lyse the cells. Then, the lysates was centrifuged at 350 × g (approx. 2000 rpm in a table-top microfuge) for 5 min at room temperature to pellet unbroken cells and tissue debris. Next, 100 µL of the resulting supernatant was centrifuged at 100,000 × g for 1 h at 37 °C to separate F-actin from soluble G-actin. Finally, the supernatant and pellet were analysed for actin content (G-actin in the supernatant versus F-actin in the pellet) by Western blot.
8
Quantifying F-actin and G-actin in cells
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F-actin and G-actin contents in a cell population were analyzed using a F-actin/G-actin assay kit, according to the manufacturer’s instructions (Cytoskeleton, Denver, CO). Briefly, the cells were treated for 3 days and lysed with prewarmed LAS2 lysis buffer. The cell lysate was centrifuged for 5 min at 350 × g to remove the cell debris, followed by ultracentrifugation at 20,000 × g for 1 h at 4 °C. The F-actin in the pellet was resuspended and incubated with 100 μL F-actin depolymerization buffer. Equal volumes of G-actin in the supernatant and F-actin fractions were mixed with 5 × SDS sample buffer and run on SDS–polyacrylamide gel electrophoresis. WB analysis was performed using rabbit anti-actin polyclonal antibody (1:500) and the intensity of each WB band was analyzed using ImageJ 1.4.3.67.
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