利用二维电泳系统分析类囊体蛋白复合物

Analysis of thylakoid protein complexes by two-dimensional electrophoretic systems.

作者信息

Sirpiö Sari, Suorsa Marjaana, Aro Eva-Mari

机构信息

Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.

出版信息

Methods Mol Biol. 2011;775:19-30. doi: 10.1007/978-1-61779-237-3_2.

DOI:10.1007/978-1-61779-237-3_2

PMID:21863436

Abstract

Photosynthetic machinery in the thylakoid membrane is prone to modifications depending on -environmental, developmental, and morphological parameters. Such plasticity in the composition of the thylakoid membrane protein complexes guarantees efficient function of the photosynthetic machinery. In this chapter, we describe methods for separation of thylakoid membrane protein complexes at high resolution by two-dimensional gel electrophoretic systems. Solubilization of the thylakoid membrane protein complexes either by dodecylmaltoside or digitonin is described first. Then, two partially overlapping -methods, blue native gel electrophoresis and high-resolution clear native gel electrophoresis, are demonstrated to separate the individual protein complexes. Finally, denaturing SDS-polyacrylamide gel electrophoresis is used to reveal the protein composition of each complex. Critical points in all protocols are addressed and representative examples of the composition of Arabidopsis thaliana thylakoid membrane protein complexes are shown.

摘要

类囊体膜中的光合机构容易根据环境、发育和形态参数发生改变。类囊体膜蛋白复合物组成的这种可塑性保证了光合机构的高效功能。在本章中，我们描述了通过二维凝胶电泳系统高分辨率分离类囊体膜蛋白复合物的方法。首先介绍了用十二烷基麦芽糖苷或洋地黄皂苷溶解类囊体膜蛋白复合物的方法。然后，展示了两种部分重叠的方法，即蓝色天然凝胶电泳和高分辨率清晰天然凝胶电泳，用于分离各个蛋白复合物。最后，使用变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳来揭示每个复合物的蛋白质组成。阐述了所有实验方案中的关键点，并展示了拟南芥类囊体膜蛋白复合物组成的代表性实例。