Any protocol to do so or any suggestions to isolate plasma membrane from HeLa cells.
Swapnil: You can surely use lectins such as ConA to isolate components of the plasma membrane and use the reporter-labeled lectin to monitor. By the way, ConA preferentially binds to alpha-mannoside containing glycoproteins which is less abundant on plasma membrane, as they were converted to complex carbohydrates with terminal sialic acids. ConA-binding proteins are more likely be found in the membranes of lysosomes.
The best way is to do sucrose density centrifugation. Lyse the cells with some kind of ball bearing homogenizer in 0.25M sucrose, and 50mM Tris, pH 8, 5mM MgCl2 plus protease inhibitor cocktail. Spin at 100,000 x g. Resuspend pellet in the 0.25m sucrose buffer without the magnesium. Layer onto a sucrose gradient from 23-43%. Put in swinging bucket for 24 hr. At 100,000 xg Remove tube. At each interface are different membrane components of the cell. One or two interfaces will be enriched with plasma membrane components. You can collect each interface and assay for plasma membrane markers. Then take the one that is enriched the most, solubilize with 1% NP40 or DDM, spin, take the cleared solution, and load onto a ConA Column to further purify.
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