Isolation of the erythrocyte membrane cytoskeleton

Principle In this procedure erythrocytes are treated with Triton X-100 which is reported to solubilize the membrane lipid leaving the underlying cytoskeletal network intact. The cyto-skeletons are separated from cytosolic components, Triton and solubilized lipid by centrifugation through a sucrose solution. The high salt concentration of the sucrose solution ensures the removal of residual lipid and integral membrane proteins from the cytoskeletal network. 

Solutions Triton X-100 extraction solution-. Triton X-100 (150 mg/ml), Hepes (24 mM), sodium chloride (0.15 M), disodium EDTA (0.5 mM), dithiothreitol (0.5 mM), pH 7.0 (prepared fresh each day) Sucrose wash Sucrose (30%, w/v), Hepes (24 mM), potassium chloride (0.6 M), ATP (0.5 mM), disodium EDTA (0.5 mM), dithiothreitol (0.5 mM), pH 7.0 (prepared fresh each day) 

Phenylmethysulphonylfluoride (PMSF) PMSF (5%, w/v) in methanol (prepared fresh each day) 

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