Lipid Profiling Extraction Method for Arabidopsis Seeds
A .pdf of this method, including shipping directions, can be found here.
NOTES: Use glass tubes with Teflon-lined screw caps. Use a Dounce-type homogenizer – ground glass to ground glass. Use glass Pasteur pipets for transferring seeds/solvent.
Volumes may be doubled for larger samples or ease of homogenizing.
1) Weigh seeds, using a microgram balance. Lyophilizing first may be best for consistent dry weight.
2) Drop seeds into 1.0 ml 75ºC isopropanol with 0.01% BHT. Heat 15 minutes. Let cool. (Hot isopropanol inactivates phospholipase enzymes.)
3) Transfer to homogenizer; homogenize thoroughly. Transfer to 17 ml glass tube for extraction. Rinse homogenizer with 1.0 ml chloroform and 1.0 ml methanol to recover all the seed parts/lipids; add to isopropanol/seed mix in tube. Add 0.8 ml water to solvent/seed mix. Shake well – should be one phase.
4) Add 1.0 ml chloroform and 1.0 ml water. Shake well. Centrifuge to split phases.
5) Remove lower layer (chloroform and lipids). SAVE to clean glass tube.
6) Add 1.0 ml chloroform, shake, and centrifuge. SAVE this lower layer in same tube as first.
7) Add 1.0 ml chloroform, shake, and centrifuge. SAVE this lower layer in same tube as above.
8) Add small amount (0.5 ml) 1 M KCl to chloroform/lipids. Shake well; centrifuge. Remove thin water layer on TOP and discard.
9) Add small amount (1.0 ml) water to chloroform/lipids. Shake well; centrifuge. Remove thin water layer on TOP and discard. (This backwash removes any proteins and carbohydrates that may have been carried through the extraction.)
10) Dry to small volume. Fill tubes with nitrogen, store at -20°C (freezer). When you are ready to prepare for shipping, evaporate completely under nitrogen gas or a speedvac, and redissolve in about 1.0 ml chloroform. Transfer to 2.0 ml clear glass vial with Teflon-lined screw cap (for example: clear glass; 2 mL; solid PTFE-lined cap; 1/2 dr., Fisher Scientific catalog #03-391-7A, Thermo Scientific No.: B7800-1). Evaporate the solvent before shipping. ALWAYS let us know before you send your samples: firstname.lastname@example.org