Abstract
For many studies regarding important chloroplast processes such as oxygenic photosynthesis, fractionation of the total chloroplast proteome is a necessary first step. Here, we describe a method for isolating the stromal, the thylakoid membrane, and the thylakoid lumen subchloroplast fractions from Arabidopsis thaliana leaf material. All three fractions can be isolated sequentially from the same plant material in a single day preparation. The isolated fractions are suitable for various proteomic analyses such as simple mapping studies or for more complex experiments such as differential expression analysis using two-dimensional difference gel electrophoresis (2D-DIGE) or mass spectrometry (MS)-based techniques. Besides this, the obtained fractions can also be used for many other purposes such as immunological assays, enzymatic activity assays, and studies of protein complexes by native-polyacrylamide gel electrophoresis (native-PAGE).
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Acknowledgments
Thomas Kieselbach for the photo shown in Fig. 2, suggestions and comments on this chapter, the Lawski foundation for postdoctoral support to Y.M., the Lars Hiertas Memorial Fund for financial support to M.H., and the Swedish Research Council for financial support to W.P.S.
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Hall, M., Mishra, Y., Schröder, W.P. (2011). Preparation of Stroma, Thylakoid Membrane, and Lumen Fractions from Arabidopsis thaliana Chloroplasts for Proteomic Analysis. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 775. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-237-3_11
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DOI: https://doi.org/10.1007/978-1-61779-237-3_11
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