Centrifuge at 12,000 RPM for 5 min. Air dry pellet for 5 min at room temp. Resuspend in a small volume and assay protein concentration with a commercially available dye-binding assay. If protein concentration is too high, adjust by adding additional Sample Buffer mix. The cytoskeleton fraction of cells contains cytoplasmic proteins such as actin, myosin and the intermediate filament proteins, as well as many plasma membrane proteins that are anchored to the cytoskeleton.
Extraction is performed based on the nonionic detergent insolubility of cytoskeletal proteins.
The following method of nonionic detergent extraction for the preparation of cytoskeletal protein samples is suitable for most cultured mammalian cells. This procedure can be used as a model for preparing proteins from any subcellular fraction. Use only reagent-grade chemicals when preparing these reagents. M 100 mL Isopropanol Mix the ingredients and store at 4 deg. Combine all reagents, then add Milli-Q water to 1.0 L. 2.