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Electron-microscopical localization of chloroplast proteins by immunogold labelling on cryo-embedded spinach leaves
Authors:Klaus Adler  Christoph Arkona  Renate Manteuffel  Karl-Heinz Süss
Institution:1. Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea;2. Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, Seoul, Republic of Korea;3. Plant Biochemistry, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany;4. Howard Hughes Medical Institute, Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102;5. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;1. Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany
Abstract:Cryo-substituted spinach leaf pieces, embedded in LR-White resin by chemical polymerization at low temperature, were used to localize enzymes within chloroplasts by immuno-electron microscopy. Employing monospecific antibodies and protein A-gold as label, the spatial distribution of six chloroplast enzymes was investigated. Statistical methods were used to determine whether each enzyme was bound to the thylakoids. By this means the coupling factor 1 (CF1), ferredoxin-NADP+ reductase, sedoheptulosebisphosphatase, and ribulose-5-phosphate kinase were found to be membrane-associated. In case of glyceraldehyde-3-phosphate dehydrogenase the label was only weak and an unequivocal determination of its location was not possible. In contrast, ribulosebisphosphate carboxylase was randomly distributed throughout the chloroplast.
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