title: Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.
abstract: Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.
authors.affiliation: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
authors.forename: Úrsula
authors.initials: Ú
authors.lastname: Flores-Pérez
authors.affiliation: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK. paul.jarvis@plants.ox.ac.uk.
authors.forename: Paul
authors.initials: P
authors.lastname: Jarvis
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D002736:Chloroplasts / Q000737:chemistry*; D003470:Culture Media / Q000737:chemistry; D007425:Intracellular Membranes / Q000737:chemistry*; D011205:Povidone / Q000737:chemistry; D036226:Seedlings / Q000737:chemistry; D012639:Seeds / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D003470:Culture Media; D013395:Sucrose; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
keywords: Arabidopsis; Chloroplast isolation; Envelope; Percoll; Stroma; Suborganellar fractionation; Sucrose step gradient; Thylakoid