Purification of Chloroplast

After deveining，100 to 500 g or freshly harvested leaves or cotyledons were homogenized in a metal waring blender with liquid nitrogen（Rhodes and Kung，19 81）.When the liquid nitrogen had evaporated and the powder had warmed to-5℃at room temperature.three weight volumes of isolation buffer containing 0.33 mol/L sorbitol.5 mmol/L MgCI2，50 mmol/L Tris-HCl（pH 8.0）.0.1%BSA and 0.1 mmol/L mercaptoethanol（Walbot，1977）were added to the powder with rapid mixing.The suspension was filtered through miracloth and centrifuged at 1.500 xg for 15 min.The crude chloroplast pellets were gently resuspended in the same buffer and puriried through a discontinous silica sol gradient（Walbot，1977）containing 0.24 mol/L sucrose throughout.(https://www.daowen.com)