Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
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Author:
Date:
2018-07-05
[Abstract] The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from Arabidopsis thaliana.
[摘要] 不稳定的类囊体蛋白复合物的分级组成和相互作用可以通过连续的2D天然凝胶电泳系统来评估。 温和的非离子洗涤剂洋地黄皂苷用于溶解不稳定的蛋白质超级和巨型复合物,然后用第一维蓝色天然聚丙烯酰胺凝胶电泳(1D-BN-PAGE)分离。 将洋地黄皂苷衍生的蛋白质复合物用更强的去污剂β-DM进一步溶解,随后在正交的2D-BN-PAGE上分离,以从较高级的超复合物中释放较小的蛋白质亚复合物。 在这里,我们描述了来自拟南芥的类囊体蛋白复合物的2D-BN-PAGE分析的详细方法。
【背景】在类囊体膜中发生光合作用的光反应,在高等植物中,由贴壁的grana类囊体和非贴壁的基质类囊体组成。光反应由多亚基蛋白复合物光系统(PS)I和II,细胞色素b 6 f和ATP酶催化。 PSII及其光捕获天线复合物(LHCII)在grana-thylakoids中最为丰富,因此在空间上与基质类囊体定位的PSI-LHCI复合物隔离(Andersson和Anderson,1980)。格拉纳和基质类囊体之间的间期在两个光系统中都得到了丰富(Albertsson,2001; Suorsa et al。,2015)。通过光依赖性LHCII和PSII蛋白的可逆磷酸化介导,光系统与LHCII一起组装成更大的超级和超级复合物。 ...
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Gradient Flotation Centrifugation of Chloroplast Membranes
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Author:
Date:
2014-09-05
[Abstract] Plastoglobules are lipoprotein particles physically attached to thylakoids in chloroplasts (Kessler et al., 1999). They are mainly composed of polar lipid, neutral lipids, and proteins (Vidi et al., 2006). Here we used simple sucrose gradient flotation centrifugation method to purify the plastoglobules from total chloroplast membranes (Vidi et al., 2007, Shanmugabalaji et al., 2013).
[摘要] 塑性球蛋白是物理附着于叶绿体中类囊体的脂蛋白颗粒(Kessler等人,1999)。 它们主要由极性脂质,中性脂质和蛋白质组成(Vidi等人,2006)。 在这里,我们使用简单的蔗糖梯度浮选离心法从总叶绿体膜中纯化质体球(Vidi等人,2007,Shanmugabalaji等人,2013)。
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