Isolation of Plastoglobules for Lipid Analyses

Plastoglobules are plastid compartments designed for the storage of neutral lipids. They share physical and structural characteristics with cytosolic lipid droplets. Hence, special care must be taken to avoid contamination by droplets during plastoglobule purification. We describe isolation pure plastoglobules from Arabidopsis thaliana leaves, methods we use determine their composition. After preparation a crude chloroplast fraction, isolated membranes two steps ultracentrifugation on discontinuous sucrose gradients. For analyses, total lipids then extracted standard chloroform–methanol protocol, polar separated liquid–liquid extraction. While classes subsequently thin-layer chromatography (TLC) classical Vitiello solvent mix, double TLC development has performed lipids, separate phytyl steryl esters. Lipids quantified gas after conversion fatty acids into methyl