Lipid Extraction from Cells
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- Wash cells 3X with PBS to remove serum.
- Remove cells by scraping cells into PBS.
- Pellet cells in ultra centrifuge; max speed for 5min.
- Add ~20X volume of 1:1 chloroform:methanol, vortex to suspend and rotate 20min at RT.
- Spin pellet max speed for 5min, recover fluid phase.
Option 1: Crude extract alone
- Dry chloroform solution from step 5 above, using inert gas (N2, CO2, argon, etc).
- Re-suspend lipids in desired media or solvent.
- Note: this solution will contain gangliosides, small polar molecules, etc.
Option 2: Lipid Purification
- Mix a 0.2 volume of PBS into the mixture produced by step 5 of “Initial extraction”, vortex several seconds to mix.
- Centrifuge 2000rpm in minifuge to separate phases. Upper (water) phase contains gangliosides, lower (chloroform) layer contains purified lipids.
- Dry the lower layer under inert gas
- Suspend as needed