title: Isolation of Arabidopsis thylakoid membranes and their use for in vitro protein insertion or transport assays.
abstract: This chapter focuses on the techniques of chloroplast isolation; their fractionation into envelopes, stroma, and thylakoids; and their further use for in vitro protein transport assays. In addition to the isolation of thylakoids, this chapter also describes the experimental steps of both protein translocation across the thylakoid membrane and protein integration into the membrane. Protein translocation and integration can be analysed by the radioactive labelling of substrate proteins using an in vitro transcription and translation system. The translocated or integrated proteins can then be detected by autoradiography. Our protocol allows the analysis of these transport systems in wild-type Arabidopsis or mutants that lack or overexpress soluble or membrane transport factors that could be of potential interest.
authors.affiliation: Molecular Biology of Plant Organelles, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bals
authors.forename: Danja
authors.initials: D
authors.lastname: Schünemann
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001345:Autoradiography; D001681:Biological Assay / Q000379:methods*; D002734:Chlorophyll / Q000378:metabolism; D061146:Chlorophyll Binding Proteins / Q000378:metabolism; D060365:Chloroplast Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018532:Pisum sativum / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D029681:Arabidopsis Proteins; D061146:Chlorophyll Binding Proteins; D060365:Chloroplast Proteins; D002734:Chlorophyll