Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography
Section snippets
Lipid extraction
Plant tissue (1–10 g fresh wt) was ground in liquid N2 to a fine powder using mortar and pestle. Cultured tobacco BY2 cells (Nicotiana tabacum bright yellow 2) or yeast (up to 2 g) was harvested by centrifugation. Phosphoinositides were extracted from ground powder or cell pellets by an acidic lipid extraction method allowing quantitative extraction of the anionic lipids [22]. Note that protonation of phosphoinositides was required for successful extraction and that no quantitative extraction was
Results and discussion
Because lipids are isolated from TLC plates after development, for quantitative purposes, it is critical to assess the rates of lipid recovery for this step. Equal amounts (10 μg) of various phosphoinositides were spotted onto silica plates (Merck) that were subsequently developed in different solvents. After the plates were developed, lipids were isolated and dried as described above. Identical amounts (10 μg) of control samples were added to fresh glass tubes and directly dried under streaming N
Acknowledgments
We thank Dr. Ivo Feussner (Göttingen University, Germany) for helpful discussion and critical reading of this article. We gratefully acknowledge financial support from the Union zur Förderung von Öl- und Proteinpflanzen e.V. (to M.H.) and an Emmy Noether grant from the German Research Foundation (DFG) (to I.H.).
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