Lipid Profiling Extraction Method for Arabidopsis Leaves (Method 1)
This procedure is generalized. Use of smaller samples is possible. Please contact Mary Roth at email@example.com (please include a subject line) to inquire.
A .pdf of this method, including shipping directions, can be found here.
Take up to 8 leaves (or up to 3 whole little plants- see NOTE #2); quickly immerse in 3 ml 75ºC (preheated) isopropanol with 0.01% BHT (butylated hydroxytoluene, e.g., Sigma B1378) for 15 min. Use a 50 ml (25 x 150 mm) glass tube with a Teflon-lined screw cap. NOTE #1: It is extremely important that the plants be extracted immediately after sampling and that the isopropanol be preheated. Plants have very active phospholipase D, which is activated upon wounding; failure to place the sampled tissue quickly into hot isopropanol will result in generation of phosphatidic acid. NOTE #2: To do the analysis, only a fraction of an Arabidopsis leaf is needed. Use of more tissue reduces variability among samples. Dry weights of 5 to 30 mg, measured as in step 7, are recommended.
Add 1.5 ml chloroform and 0.6 ml water, vortex; then agitate (shaking incubator) at room temperature for 1 hour. Transfer (long, glass Pasteur pipettes) lipid extracts to glass tubes with Teflon-lined screw-caps.
Add 4 ml chloroform/methanol (2:1) with 0.01% BHT; shake 30 min. Repeat this extraction procedure on all samples until the leaves of every sample become white, but typically each sample is extracted the same number of times. (Use one pipette in each sample for all extractions, leaving them in the removed extract while extracting the remaining materials.) It's OK to leave for somewhat longer than 30 min on later extractions. Usually you will need about 5 extractions, including the one with the isopropanol.
Add 1 ml 1 M KCl to the combined extract, vortex or shake, centrifuge, discard upper phase.
Add 2 ml water, vortex or shake, centrifuge, discard upper phase.
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). ALWAYS let us know before you send your samples: firstname.lastname@example.org
Dry extracted leaves at 105°C oven overnight; weigh for "dry" weights.
Note: This procedure can be adapted for other plant tissues, including roots, stems, flowers, and siliques.
Lipid Profiling Extraction Method for Leaves (Method 2, tested on Arabidopsis and sorghum)
A more rapid method of lipid extraction was recently described by Shiva et al. (2018): https://plantmethods.biomedcentral.com/articles/10.1186/s13007-018-0282-y
This approach gets a good extraction from leaves in a single step. If following Method 2, after removing the plant materials (as shown in Figure 2), follow steps #6 and #7 in the above protocol.