title: Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings.
abstract: The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.
authors.affiliation: The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Zeng
authors.affiliation: The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia.|Department of Biochemistry, University of Cambridge, Cambridge CB2 1QR, UK.
authors.forename: Berit
authors.initials: B
authors.lastname: Ebert
authors.affiliation: Joint BioEnergy Institute and Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94702, USA.
authors.forename: Harriet T
authors.initials: HT
authors.lastname: Parsons
authors.affiliation: The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia.|Department of Biochemistry, University of Cambridge, Cambridge CB2 1QR, UK.
authors.forename: Carsten
authors.initials: C
authors.lastname: Rautengarten
authors.affiliation: The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Antony
authors.initials: A
authors.lastname: Bacic
authors.affiliation: Joint BioEnergy Institute and Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94702, USA. jheazlewood@unimelb.edu.au.|School of BioSciences, The University of Melbourne, Melbourne, 3010, VIC, Australia. jheazlewood@unimelb.edu.au.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D018274:Electroporation / Q000379:methods; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry; D006056:Golgi Apparatus / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D036226:Seedlings / Q000737:chemistry* / Q000254:growth & development; D012639:Seeds / Q000254:growth & development; D013395:Sucrose / Q000737:chemistry; D014170:Transformation, Genetic; D014908:Triticum / Q000737:chemistry* / Q000254:growth & development; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D005456:Fluorescent Dyes; D010940:Plant Proteins; D013395:Sucrose; D017766:Acid Anhydride Hydrolases; C023837:nucleoside-diphosphatase
keywords: Density gradient centrifugation; Golgi apparatus; Nucleoside diphosphatase; Subcellular localization