The Plant Endoplasmic Reticulum by Chris Hawes & Verena Kriechbaumer

Author:Chris Hawes & Verena Kriechbaumer
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

3.6 Ceramide and Glucosylceramide Analysis by HPTLC and GC-MS Analysis of Their Fatty Acids

Under the conditions of polar lipid (phospholipids) separation described above, glucosylceramides and sterylglucosides do not separate. These lipids are separated on HPTLC plates using the solvent system chloroform/methanol (85/15, v/v) [10]. In this elution system, ceramides can also be isolated on HPTLC plates. Quantification by densitometry using ceramide and glucosylceramide standards (10 μg per standard) can be performed as explained for phospholipids in 3.2.

For acyl-chain characterization of ceramides and glucosylceramides by GC-MS, scraped lipid bands (after iodine vapor revelation, see Subheading 3.2) are directly incubated with 1 ml of 5% sulfuric acid solution in methanol (implemented with standards: 5 μg/ml of C17:0 and 5 μg/ml of h14:0) for transesterification which is made overnight at 85 °C to produce FAMEs. Those are then washed by adding 1 ml of NaCl 2.5% and 1 ml of hexane 99%. After vigorous shaking and centrifugation at 700 × g for 5 min at room temperature, the higher phase is harvested, placed in a new tube and buffered with 1 ml of 100 mM Tris, 0.09% NaCl, pH 8 with HCl. After vigorous shaking and centrifugation at 700 × g for 5 min at room temperature, the higher phase is harvested, placed in a new tube and evaporated with needles evaporating pan. Then, 200 μl of N,OBis(trimethylsilyl)trifluoroacetamide + 1% trimethylsilyl (BSTFA + 1% TMCS) are added and incubated at 110 °C for 20 min. After evaporation, FAMEs are resuspended in 100 μl of 99% hexane and run on GC-MS with the same program described in Subheading 3.3.

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