Isolation of Lipids from Human Blood-Derived Neutrophils by Biphasic Separation
Trascrizione
Take a glass tube containing neutrophil homogenate with released intracellular components.
Add methanol, a polar solvent, that disrupts lipid-lipid and lipid-protein interactions. The inherent polarity of methanol evenly distributes polar lipids, solubilizing them. Next, add chloroform, a nonpolar organic solvent, that interacts with nonpolar lipids, causing them to disperse and solubilize.
Centrifuge to separate the protein-containing pellet from the supernatant containing lipids, polysaccharides, and cell debris. Transfer the supernatant to a fresh glass tube. Add chloroform and distilled water. Centrifuge the sample.
Water addition spontaneously forms a biphasic system, partitioning biomolecules within the lower chloroform and the upper water-methanol layers.
The chloroform layer comprises dissolved nonpolar lipids, while the upper layer contains non-lipid contaminants and more polar lipids. Remove the upper water-methanol layer without disturbing the lipid-containing layer.
Use a vacuum concentrator to remove the solvent from the lipids. Store at lower temperatures until further use.
