A METHOD FOR SIMULTANEOUS ISOLATION OF CHLOROPLASTS AND MITOCHONDRIA FROM ICE PLANT

This research work provides a detailed method for the simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of the halophyte model Ice plants collected at the same time of the same biological samples and their protein extraction. The protocol includes three main steps: (1) Simultaneous isolation of pure, intact chloroplasts and mitochondria; (2) Chloroplasts and mitochondrial protein extraction and purification; and (3) Chloroplasts and mitochondrial protein determination by EZQ kit. The modifications in this method are: Using a Polytron homogeniser to disrupt and release of organelles and prepare Percoll gradient with a Masterflex pump. The protease inhibitor cocktail plus 1 μM Leupeptin and 1 μM E-64 were added to the grinding and protein extraction buffer to control the activity of plant-specific proteases and minimise protein degradation within the samples. The chloroplast and mitochondrial protein concentration was determined using an EZQ™ Protein Quantification Kit (R33200) from Molecular Probes™ according to the manufacturer's instructions.