Lipid Extraction Method for Arabidopsis (and other) 1xbet best casino website (Method 1)
This procedure is generalized. Use of smaller 1xbet best casino website is possible. This procedure can be adapted for other plant tissues, including roots, stems, flowers, and siliques. Please contact Mary Roth at mrroth@ksu.edu (please include a subject line) to inquire.
A .pdf of this method, including 1xbet best casino website directions, can be found here.
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Take 1 to 8 leaves (or up to 3 whole little plants- see NOTE #2); quickly immerse in 3 1xbet best casino website 75ºC (preheated) isopropanol with 0.01% BHT (butylated hydroxytoluene, e.g., Sigma B1378) and continue to heat for 15 1xbet best casino website . Use a 50 1xbet best casino website (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; use of smaller amounts of tissue can provide information about individual plants and plant tissues. Dry weights of 5 to 30 mg, measured as in step 6, are recommended.
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Add 1.5 1xbet best casino website chloroform and 0.6 1xbet best casino website water, vortex; then agitate (shaking incubator) at room temperature for 1 hour. Transfer (long, glass Pasteur pipettes) 1xbet best casino website extracts to glass tubes with Teflon-lined screw-caps.
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Add 4 1xbet best casino website 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 be sure to extract each sample 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 the tubes shaking for somewhat longer than 30 min on later extractions, and leaving one of the extractions shaking overnight is a good idea for difficult-to-extract tissues. Usually you will need about 5 extractions, including the one with the isopropanol.
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Optional back-washes: Add 1 1xbet best casino website 1 M KCl to the combined extract, vortex or shake, centrifuge, discard upper phase. Add 2 1xbet best casino website water, vortex or shake, centrifuge, discard upper phase. These backwashes will yield a cleaner lipid sample, but small amounts of the more polar lipids, such as lysolipids, will be lost.
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Fill tubes containing lipid extract with nitrogen, store at -20°C (freezer). When you are ready to prepare for shipping, evaporate solvent completely under nitrogen gas or in a speedvac, and redissolve in about 1.0 1xbet best casino website chloroform. Transfer to 2.0 1xbet best casino website clear glass vial with Teflon-lined screw cap (for example: clear glass; 2 1xbet best casino website ; solid PTFE-lined cap; 1/2 dr., Fisher Scientific catalog #03-391-7A, Thermo Scientific No.: B7800-1). Evaporate solvent from the samples in the 2-1xbet best casino website vials before shipping. ALWAYS let us know before 1xbet best casino website send your samples: mrroth@ksu.edu
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Dry extracted 1xbet best casino website at 105°C oven overnight; weigh for "dry" weights preferably using a balance that weighs (in grams) to 6 decimal places (i.e., micrograms).
Video demonstration 1xbet best casino website of Method 1:
Click here to watch at YouTube (full screen available)
Lipid Extraction Method for 1xbet best casino website (Method 2, tested on Arabidopsis and sorghum)
A more rapid method of 1xbet best casino website 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 1xbet best casino website in a single step. If following Method 2, after separating the plant materials from the lipid extract, follow steps #5 and #6 in the above protocol.
video 1xbet best casino website demonstration of Method 2:
Click here to watch at YouTube (full screen available)
Lipid Extraction Method for 1xbet best casino website (Method 3 - if you need GIPC analysis, in addition to that of other lipids)
If you want to include glycosylinositolphosphoceramide (GIPC) in your extract, you need to do additional extractions of your 1xbet best casino website with "Solvent H".
1xbet best casino website H from Markham et al. (2006, http://www.jbc.org/content/281/32/22684.full.html):
1xbet best casino website /hexane/water 55:20:25, upper phase discarded
440 1xbet best casino website 2-propanol w/0.01% BHT (0.04g)
160 1xbet best casino website hexane (HPLC grade)
200 1xbet best casino website water
Mix these together (stirring vigorously), and remove the upper phase for discard. Lower phase should be clear and is 1xbet best casino website H.
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Complete either steps 1-3 in Method 1 or the Method 2 1xbet best casino website -step method.
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To the remaining leaf tissue, add 4 1xbet best casino website “Solvent H”. Put tube on heating block at 60°C for 15 min. Remove solvent and combine with the chloroform/methanol lipids extracted previously.
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Repeat three more times for a total of 4 “Solvent H” extractions. The total volume of all extracted lipids plus solvents is about 37 1xbet best casino website (if you used Method 1).
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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, at less than 40ºC, and redissolve in about 1.0 1xbet best casino website chloroform. Transfer to 2.0 1xbet best casino website clear glass vial with Teflon-lined screw cap (for example: clear glass; 2 1xbet best casino website ; solid PTFE-lined cap; 1/2 dr., Fisher Scientific catalog #03-391-7A, Thermo Scientific No.: B7800-1). Evaporate solvent from the samples in the 2-1xbet best casino website vials before shipping. ALWAYS let us know before 1xbet best casino website send your samples: mrroth@ksu.edu
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1xbet best casino website extracted with Solvent H 1xbet best casino website stick to the glass when dry, unless a bit of chloroform is added and the 1xbet best casino website are pushed into a ball. A spatula works well for forming the ball. Dry extracted 1xbet best casino website at 105°C in an oven overnight, and weigh to 6 decimal places (0.000000g) for dry extracted tissue weight. (Just dump the dried tissue into a weigh boat.)