boiling-water-protocol

 

Use glass tubes  with screw caps and teflon septa to avoid contamination of the lipid extract. It is a good idea to rinse glass tubes with chloroform before use to remove any contamination. Never use rubber septa in the caps! It is best to use fresh leaves directly taken from plant, not frozen leaves.

 

1. Cut material (leaf or roots etc., ca. 100 mg fresh weight) fresh from plant. It is best to skip fresh weight measurements and determine dry weight later because phospholipase D action might degrade your phospholipids (see point 9). Work fast at this point and immediately after cutting

 

2. Put material into glass tube (wide opening, can also use small beaker or flask covered with aluminium foil) containing boiling water  for 20 min standing in 100°C water bath. This destroys all lipase activity. If you use a tube with screw cap, make sure that it has a teflon septum.

 

3. Take the leaf/leaves out of the water and transfer it in a new tube (12 x 100 mm)

 

4. Extract leaf with 1 ml CHCl3:MeOH 1:2, vortex carefully and harvest all (!) organic lipid extract (no phase separation) into a new vial.

 

5. Extract leaf with 1 ml CHCl3:MeOH 2:1, vortex carefully and harvest all (!) lipid extract (no phase separation) and combine it with the first extract. Make sure that leaf is white (not green) after second extraction. If the leaf is not white, do another extraction with 1 ml CHCl3.
It is important to extract all lipids at this step to be quantitative.

 

6. To the combined  fractions, add 1 ml chloroform (if a third extraction has been done this is not necessary) and 0.75 ml 300 mM NH4OAc solution, such that the final ratio of CHCl3:MeOH: NH4OAc-solution is: 2:1:0.75.

 

7. vortex, centrifuge, harvest lower (lipid) phase; take out all (!) of the chloroform phase, but avoid taking the aqueous phase

 

8. At this point, you can send samples (upright, in glass tubes with screw caps with teflon septa) at room temperature by normal mail for lipid quantification. You can also blow off the liquid with a sample concentrator with nitrogen gas and fill the tube with nitrogen gas (to avoid oxydation of double bonds by O2 from the air). Then close the tube with cap and ship the dry lipid samples to us. For shipment via airmail this is better because no solvents are shipped.

 

9. Determine the dry weight (after lipid extraction) of the leaf. To this end, put the leaf residue (in glass vial) into oven at > 100 °C overnight. Next morning, determine dry weight of samples

 

 

In Bonn, we will continue like this:

 

 

10. if shipped in solvent: evaporate solvent from lipid extract with air or N2 gas

 

11. Samples are re-dissolved in 1 ml Q-TOF or Q-Trap solvent for direct infusion on Q-ToF or on Q-Trap, respectively.

 

12. For phospho- and galactolipidmeasurement: prepare an exact dilution (e.g. 1:10) in Q-TOF solvent plus  internal standard mix.
(for example 10 µl extract + 80 µl Q-ToF solvent + 10 µl IS. Can store the lipid extract at -