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Acidic Chloroform/Methanol Protocol

Isolation of glycerolipids (phospholipids, galactolipids, triacylglycerol) using acidic chloroform-methanol extraction for mass spectrometry

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Please make sure that your samples are free of detergents, PEG, plasticizers etc. We recommend that you extract your samples at your institution and ship them to us at ambient temperature.

Use this protocol for thick plant tissues, e.g. roots (Lotus, Medicago,..).

  1. Take out roots from soil/sand and wash off dirt with water.
  2. Dry on paper towel.
  3. Cut out root segment (50-100 mg) with scissors/scalpel or razor blade (e.g. mycorrhized root segment). Be fast, because cutting/wounding causes induction of phospholipases.
  4. Transfer root to 2 ml microfuge tube (as fast as possible) (snap cap tube, if possible determine weight of empty microfuge tube before) (do not use screw cap with rubber seal) and freeze in liquid nitrogen (important to be fast at this point because cutting/wounding causes lipid degradation).
  5. Determine weight of microfuge tube plus root (frozen) on balance quickly without thawing. Subtract the weight of the empty tube determined before. This would give the fresh weight.
    Store at -70. You can ship samples to Bonn on dry ice at this point, or continue.
  6. Grind the sample (frozen) with Precellys® homogenizer and ceramic beads. Add 0.2 ml chloroform/methanol/formic acid (1:1:0.1) and homogenize once again.
  7. Add 0.4 ml chloroform/methanol/formic acid (1:1:0.1, v/v) and 0.2 ml aqueous 0.2 M H3PO4, 1 M KCl to the frozen tissue (final ratio organic solvents/aqueous portion 2:1).
  8. Vortex thoroughly, centrifuge microfuge tube for 5 min at 5000 rpm to get phase separation.
  9. Transfer (carefully) bottom phase (chloroform) which contains the lipids to a new glass tube with screw cap (Teflon septum).
  10. Add 0.4 ml chloroform to the tissue, vortex and centrifuge again. Repeat this step a third time.
  11. Harvest bottom phase (chloroform) and combine the three chloroform phases.
  12. Ship lipid extract to Bonn.

Use this protocol for animal tissues:

  1. Harvest tissue, eventually wash with PBS buffer to remove remaining liquids (e.g. blood). Transfer sample to 2 ml microfuge tube and freeze in liquid nitrogen. The sample can be stored at this point at -70°C
  2. Add 0.4 ml aqueous 0.2 M H3PO4, 1 M KCl per 100 mg wet weight (!) of frozen tissue.
  3. Homogenize the sample with Precellys® homogenizer and ceramic beads.
  4. Add 0.8 ml chloroform/methanol/formic acid (1:1:0.1, v/v).
  5. Vortex thoroughly, centrifuge microfuge tube for 5 min at 5000 rpm to get phase separation.
  6. Transfer (carefully) bottom phase (chloroform) which contains the lipids to new glass tube with screw cap (teflon septum).
  7. Add 1 ml chloroform/methanol (2:1, v/v) to the tissue, vortex and centrifuge again.
  8. Harvest bottom phase (chloroform) and combine the two chloroform phases.
  9. Ship lipid extract to Bonn.

Reference:
Hajra A (1974) On extraction of acyl and alkyl dihydroxyacetone phosphate from incubation mixtures. Lipids 9: 502-505