Summary
A major area of research in the postgenomic era has been the proteomic analysis of various subcellular and suborganellar compartments. The success of these studies is to a large extent dependent upon efficient protocols for the preparation of highly pure organelles or suborganellar components. Here we describe a simple, rapid, and low-cost method for isolating a high yield of Arabidopsis chloroplasts. The method can readily be applied to wild-type plants and different mutants, and at different developmental stages ranging from 10-day-old seedlings to rosette leaves from older plants. The isolated chloroplast fraction is highly pure, with immunologically undetectable contamination from other cellular organelles. Chloroplasts isolated using the method described here have been successfully used for proteomic analysis, as well as in studies on chloroplast protein import and other aspects of chloroplast biology.
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Acknowledgments
We thank Ramesh Patel for technical assistance, and Natalie Allcock and Stefan Hyman (Electron Microscope Laboratory, Faculty of Medicine and Biological Sciences, University of Leicester) for transmission electron microscopy. We are grateful to Sabina Kovacheva and Ramesh Patel for their helpful comments on the manuscript. We thank Marc Boutry (PMA2 H+-ATPase), Karl-Josef Dietz (PrxII F), Richard Trelease (catalase), Peter Shaw (histone H3), Henrik Scheller (PSI-D), and Kenton Ko (SSU) for generously providing antibodies. This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Genomic Arabidopsis Resource Network (GARNet), by the Royal Society Rosenheim Research Fellowship (to P.J.), and by BBSRC Grants 91/P12928 and BBS/B/03629 (to P.J.).
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Kubis, S.E., Lilley, K.S., Jarvis, P. (2008). Isolation and Preparation of Chloroplasts from Arabidopsis thaliana Plants. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 425. Humana Press. https://doi.org/10.1007/978-1-60327-210-0_16
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DOI: https://doi.org/10.1007/978-1-60327-210-0_16
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