Lipid Extraction from Cells

From Heit Lab Wiki
Jump to: navigation, search

Contents

Protocol

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