UNIT 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic Centrifugation Daphn´ e Seigneurin-Berny, 1 Daniel Salvi, 1 Jacques Joyard, 1 and Norbert Rolland 1 1 Laboratoire de Physiologie Cellulaire V´ eg´ etale, CNRS and Universit´ e Joseph Fourier, Grenoble, France ABSTRACT Chloroplasts are plant-specific organelles. They are the site of photosynthesis but also of many other essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments. This unit describes the isolation and purification of chloroplasts from Arabidopsis and spinach leaves. Differential centrifugation is first used to obtain a suspension enriched in chloroplasts (crude chloroplasts extract). In a second step, Percoll density gradient centrifugation is used to recover pure and intact chloroplasts. The Basic Protocol describes the purification of chloroplasts from Arabidopsis leaves. This small flowering plant is now widely used as a model organism in plant biology as it offers important advantages for basic research in genetics and molecular biology. The Alternate Protocol describes the purification of chloroplasts from spinach leaves. Spinach, easily available all through the year, remains a model of choice for the large- scale preparation of pure chloroplasts with a high degree of intactness. Curr. Protoc. Cell Biol. 40:3.30.1-3.30.14. C  2008 by John Wiley & Sons, Inc. Keywords: chloroplast  Arabidopsis  spinach  purification  leaves INTRODUCTION Plastids are semiautonomous organelles found in plants and some protists. In plant leaves, plastids are photosynthetically active and named chloroplasts. They are also the site of essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments (Wise and Hoober, 2006). Biochemical, physiological, and proteomic analyses of the chloroplast can only be achieved using pure and intact chloroplasts that have con- served their metabolic activities and functional surrounding envelope membranes (Block et al., 2007). Localized at the interface between the plastid stroma and the cytosol, the envelope membranes have to remain intact to allow, for instance, physiological charac- terization of the various transport systems regulating plastid metabolism. Therefore, the isolation of pure and intact organelles becomes essential when characterization of plastid functions is expected. The procedure described here couples differential centrifugation to obtain a crude extract of chloroplasts with isopycnic centrifugation (Percoll-based density gradients) to recover pure and intact chloroplasts. The Basic Protocol describes the step-by-step purification procedure to isolate chloro- plasts from Arabidopsis leaves. Since the complete sequencing of its genome (The AGI, 2000), Arabidopsis thaliana, a small flowering plant, has become a widely used model organism in plant biology. The generation of large collections of insertion mutants, to- gether with growing numbers of gene expression databases, offer important advantages for basic research in genetics and molecular biology. This Basic Protocol, to isolate chloroplasts from Arabidopsis thaliana, is provided with some tricks to optimize yield and purity. Conditions to grow Arabidopsis plants suitable for chloroplast isolation are also described in the Support Protocol. Current Protocols in Cell Biology 3.30.1-3.30.14, September 2008 Published online September 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471143030.cb0330s40 Copyright C  2008 John Wiley & Sons, Inc. Subcellular Fractionation and Isolation of Organelles 3.30.1 Supplement 40