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).
