Hall Michael, Mishra Yogesh, Schröder Wolfgang P
Department of Biological Chemistry, Institute of Chemistry and Umeå Plant Science Centre (UPSC), Umeå University, Umeå, Sweden.
Methods Mol Biol. 2011;775:207-22. doi: 10.1007/978-1-61779-237-3_11.
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).
对于许多关于诸如光合放氧等重要叶绿体过程的研究而言，对整个叶绿体蛋白质组进行分级分离是必要的第一步。在此，我们描述一种从拟南芥叶片材料中分离叶绿体基质、类囊体膜和类囊体腔亚叶绿体组分的方法。所有这三个组分可在一天的制备过程中从同一植物材料中依次分离出来。分离得到的组分适用于各种蛋白质组学分析，如简单的图谱研究，或更复杂的实验，如使用二维差异凝胶电泳（2D-DIGE）或基于质谱（MS）的技术进行差异表达分析。除此之外，所获得的组分还可用于许多其他目的，如免疫测定、酶活性测定以及通过非变性聚丙烯酰胺凝胶电泳（非变性PAGE）对蛋白质复合物进行研究。
