Lipid Profiling Extraction Method for Arabidopsis Leaves
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 in freezer; and transport to the KLRC Analytical Laboratory on dry ice. Alternatively, if you need to ship the samples, evaporate the solvent, fill the tubes to nitrogen, and ship on dry ice. You may wish to consider transferring the samples to 2 ml vials with teflon-lined caps for shipping. Please contact Mary Roth at firstname.lastname@example.org before shipping.
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.