The overall goal of this procedure is to determine the polar lipid composition of arabidopsis seedlings. This is accomplished by first extracting lipids from the seedlings. The polar lipids are then separated by thin layer chromatography.
Next specific lipid spots are isolated from the thin layer plate and converted to fatty assal methyl esters by transesterification. The final step of the procedure is to quantify the composition of fatty assal methyl esters by gas liquid chromatography. Ultimately, results can be obtained that show the molar ratio and the fatty acid profile of each polar lipid in wild type and mutant.
Hi, my name is Christophe Benning. Many of you have asked us how to do a quantitative analysis of polar lipids. We use a very simple and robust method that served us well during large scale screening of mutants in the model plant opsis or the RG kilometer motors.
Other excellent methods such as mass spectrometry based lipid profiling require expensive equipment and lots of experience and training. We use a two stage procedure that involves simple thin layer chromatography combined with quantitation of fatty acid methyl esters following the isolation of mutants. Usually a more quantitative analysis is required.
This is done by isolation of membrane lipids from the thin layer plate and preparation of fatty acid methyl esters, which are quantified by liquid gas chromatography. The method is highly reproducible and results are comparable with more complex mass spectrometry based techniques demonstrating the procedure will be Zen Wang, A graduate student from my laboratory Begin lipid extraction by harvesting 30 milligrams of four week old Arabidopsis leaves from plants grown on aga solidified medium or soil. Transfer the leaves into 1.5 milliliter propylene reaction tubes.
Add 300 microliters of extraction solvent composed of methanol, chloroform, and formic acid to each sample. Shake the samples vigorously for five minutes. Next, add 150 microliters of 0.2 molar phosphoric acid, one molar potassium chloride, and vortex.
Briefly centrifuge the sample at 13, 000 times G for one minute. At room temperature lipids dissolved in the lower chloroform phase will be spotted onto thin layer chromatography plates. To prepare TLC plates submerge a 20 by 20 centimeter silica gel coated TLC plate with loading strip into 0.15 molar ammonium sulfate solution.
After submerging for 30 seconds, dry the plate for at least two days in a covered container. During activation, the sublimation of ammonium leaves behind sulfuric acid, which protonate phosphate ar glycerol necessary for its separation from other glycerol lipids. On the day of the experiment, activate TLC plates by baking them in an oven at 120 degrees Celsius for two and a half hours.
After cooling down the activated plates to room temperature, use a pencil to draw a straight line across the plate at the origin of the chromatogram. In a fume hood, tigon tubing connected to the regulator of the nitrogen tank is used to provide a stream of nitrogen gas, slowly deliver three times 20 microliters of the lipid extract from the lower chloroform phase to the plate using a 20 microliter per pet with a 200 microliter yellow plastic tip under a slow stream of nitrogen gas. A 20 microliter per pet is used to keep the spot smaller than one centimeter in diameter.
Each plate can hold up to 10 samples as the lipid spots completely dry. In the fume hood, prepare the developing solvent composed of acetone, toluene, and water. Pour 80 milliliters of the developing solvent into a sealable TLC developing chamber and place the plate into the tank.
With the sample end facing down seal the tank. Using the clamp, the solvent will ascend the plate and lipids will be separated. The development time is approximately 50 minutes at room temperature.
When the solvent front has reached one centimeter from the top of the plate, carefully remove the plate from the tank, dry the plate completely in the fume hood for 10 minutes. Lipids separated by TLC can be either reversibly stained briefly with iodine for quantitative analysis or irreversibly stained with sulfuric acid or alpha natal. To irreversibly, stain the plate through sulfuric acid charring.
Spray the plate with 50%sulfuric acid in water in a glass spray bottle in the fume hood and bake it 120 degrees Celsius for 15 minutes. Conversely, when alpha natal staining for glycolipids, spray the plate with 2.4%weight over volume alpha natal in 10%volume over volume, sulfuric acid, 80%volume over volume. Ethanol bake the plate at 120 degrees Celsius for three to five minutes until glycolipid spots are stained pink or purple.
Perform iodine staining In a fume hood, prepare a closed LC tank with iodine crystals in a tray on the bottom of the tank leading to saturation of the atmosphere with iodine vapor. Place the TLC plate into the tank to expose it to iodine until lipids are visible. Don't expose the iodine too long as the iodine may covalently.
Modify polyunsaturated fatty acids. Remove the silica surrounding identified lipid spots from the TLC plate with the razor blade and scrape the lipid containing silica. Using a funnel, transfer the silica powder into a glass tube with a Teflon line screw cap.
Add one milliliter of one an hydrochloric acid in anhydrous methanol to each sample by glass peppe. Then add 100 microliters of 50 micrograms per milliliter penedes acid to each sample as an internal standard using a 200 microliter per pet with a 200 microliter yellow plastic tip. Also prepare a tube with only pent decanoic acid in methodol hydrochloric acid as a control.
Close the glass tubes tightly with Teflon lined caps. Incubate the glass tubes in an 80 degrees Celsius water bath for 25 minutes. Tubes must be sealed so that the solvent does not evaporate after tubes have cooled down.
Add one milliliter of 0.9%sodium chloride followed by one milliliter of hexane, and then vortex vigorously centrifuge the samples at 1000 times G for three minutes following centrifugation, remove the hexane upper layer of the sample with a pasta peppe in the fume hood and place it into a new 13 by 100 millimeter glass tube. Evaporate the hexane under a slow stream of nitrogen without drying completely dissolve the resulting fatty asal methyl esters in 60 microliters of hexane. Transfer the samples into autos sampler vials, then cap tightly.
Samples can be stored at four degrees Celsius for short term and minus 20 degrees Celsius for a few days before beginning.GLC. Ensure that the helium, hydrogen and air bottles are filled sufficient hexane must be added to the solvent reservoir and the waste container must be empty for fatty assal methyl Lester separation. Attach a DB 23 column to the machine.
Place the vials into the autos sampler. Start the chem station software for GLC on the system.Computer. Set the inlet temperature at 250 degrees Celsius with a helium flow rate at 48.6 milliliters per minute, and a pressure of 21.93 PSI.
The split ratio is 30 to one. The oven temperature is set initially at 140 degrees Celsius for two minutes and raised to 160 degrees Celsius at a rate of 25 degrees Celsius per minute. Then set the temperature to increase from 160 degrees Celsius to 250 degrees Celsius at a rate of eight degrees Celsius per minute set to hold the temperature at 250 degrees Celsius for four minutes, followed by a decrease to 140 degrees Celsius at a rate of 38 degrees Celsius per minute.
Using this temperature profile, one run takes about 21 minutes. The temperature of the flame ionization detector is 270 degrees Celsius with a hydrogen flow rate of 30 milliliters per minute. Airflow rate at 400 milliliters per minute and helium flow rate at 30 milliliters per minute.
Enter the number of vials and sample names in the run sequence table. Set the 10 microliter injector to inject two microliters of the sample per vial. When the instrument is ready, initiate the run sequence.
Examples of staining of TLC separated lipids from four week old Arabidopsis seedlings are shown here. The sulfuric acid stained lipids are charred and appear as brown spots. Alpha natal is preferred to stain glycolipids such as M-G-D-G-D-G-D-G and SQ dg.
Glycolipids stained with alpha natal carry a pink purple color while other polar lipids stain yellow. The iodine staining is reversible and gives lipids a yellowish color that will disappear over a short time as the iodine evaporates. Briefly, iodine stained lipids can be subjected to GLC analysis, although unstained lipids are preferable to reduce the breakdown of lipids if done successfully.
Distinctive signals representing different fatty asal methyl lesters will be observed after GLC fatty asal methyl esters with shorter carbon chains and fewer double bonds have shorter retention time. Using the DB 23 column. Fatty assal methyl ester profiling is a sensitive tool to identify mutants with altered lipid composition.
The MGDG 18 three fatty acid molar ratio is decreased in the TGD four dash one mutant as compared to the wild type by dividing the moles of fatty assal methyl ester for one lipid class with moles of fatty assal methyl eta. Of all lipid classes, the molar ratios of each lipid are calculated the resulting molar ratios of each lipid class from both the wild type and the mutant can be compared here. It is evident that the TGD four dash one mutant has increased relative amounts of MGDG and pg, but decreased amounts of D-G-D-G-N-P-E Thin layer chromatography allows the examination of many samples in parallel and permits a visual examination of major differences in lipid profiles.
Furthermore, looking at A TLC, you are essentially examining many enzymes and genes required for the synthesis of the lipids and therefore the lipid profile. After watching this video, you should have a good understanding of how to analyze the composition of polar lipids by TOC and GOC. Thanks for watching the video, and good luck with your experiment.
