G actin f actin in vivo assay biochem kit

A G-Actin/F-actin in vivo Assay Biochem Kit (Cytoskeleton Inc., Denver, CO, USA; BK037) was used to evaluate the G-Actin/F-actin ratio of the cochlea. G-actin and F-actin proteins were separated according to the instructions. In brief, the membranous labyrinths of the cochleae were dissected in 0.01 M PBS. Tissues were frozen in liquid nitrogen and ground into a powder. LAS2 buffer (1 ml per 0.1 g of tissue) was added to the sample, then F-actin Enhancing Solution was added to the sample at a volume ratio of 1:100. The tissue was lysed in the mixed solution for 10 min at 37°C, then the lysate was placed in a centrifuge and centrifuged at 2,000 rpm for 5 min to remove impurities after lysis. The supernatants were then pipetted into clearly-labeled ultracentrifuge tubes, and centrifuged at 100,000× g, 37°C for 1 h. The supernatant, which contained G-actin, was separated and the F-actin depolymerization buffer was added to the precipitate. The sample was placed on ice for 1 h, inverting the sample every 15 min, to obtain the G-actin solution produced by the depolymerization of F-actin. Finally, 5× SDS sample buffer was added to each tube, and western blotting was used to evaluate the G-actin/F-actin ratio.

Monomeric and filamentous actin populations in murine cardiomyocytes were separated by using G-Actin/F-actin In Vivo Assay Biochem Kit (Cytoskeleton, Inc.) according to manufacturer’s recommendations. Briefly, 5 days after plating cells were washed twice with 1×PBS and scraped off from gelatin-coated (2%) 35 mm plates in Lysis and F-actin Stabilization Buffer prewarmed to 37 °C. The solubilized lysates were incubated at 37 °C, 10 min and cell debris was clarified by centrifugation at 350 × g, 5 min. G-actin in the supernatants and F-actin in the pellets were fractionated by ultracentrifugation at 100,000 × g, 37 °C, 1 h. The pellets were solubilized in F-actin Depolymerizing Buffer on ice, 1 h by triturating with pipetting every 15 min. Supernatant and pellet fractions were resolved on SDS–PAGE, and actin in each fraction was detected by immunoblotting.
The lysis conditions were confirmed by treating the cells 4 days after plating with 0.01% DMSO (control), 2 μM Lat A (Invitrogen), or 0.15 μM jasplakinolide (Santa Cruz Biotechnology) overnight, at 37 °C and harvesting the lysates next day as described above.