| Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
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Author:
Date:
2016-07-20
[Abstract] The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
[摘要] 质体编码聚合酶（PEP）代表成熟叶绿体中的主要转录机制（Liere等人，2011; Zhelyazkova等人，2012）。 这种多亚基复合物的正确装配对于植物生长和发育是重要的（Pfalz和Pfannschmidt，2013）。 PEP聚合酶可以从可溶性和膜结合（也称为转录活性染色体，TAC）级分中纯化。 蓝色使用天然聚丙烯酰胺凝胶电泳（BN-PAGE）和蔗糖梯度沉淀，随后进行免疫印迹分析来检测PEP复合物组装体的状态。
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| Localization and Topology of Thylakoid Membrane Proteins in Land Plants
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Author:
Date:
2014-12-20
[Abstract] Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this ...
[摘要] 类囊体是在蓝细菌，藻类和植物中发现的扁平膜囊泡和蛋白质复合物的形成。在陆地植物的叶绿体中，类囊体膜系统形成称为颗粒薄片的密集堆叠的网络，其通过未堆叠的基质薄片连接。光系统II主要定位在贴壁的颗粒区域，而光系统I和ATP合酶复合物富集基质层。细胞色素e / f 复合物横向分布在堆叠和未堆叠的膜区域。光合复合物由整合蛋白和外周蛋白组成。本协议（A）的第一部分显示如何分裂类囊体成grana和基质层。该方案的第二部分（B）显示了如何区分强疏水整合膜缔合和弱静电膜和/或膜复合物缔合。由于必须特异性检测级分中的目标蛋白，针对目的蛋白产生的特异性抗体或表达标记融合蛋白的结构组分的互补无效突变体将是非常有利的。本协议（C）的最后一部分显示，如何调查内在和外围蛋白的拓扑。该方法需要针对目标蛋白的特异性抗体。对于内在膜蛋白，需要肽特异性抗体或表位标记的形式。该方案适合于低于5kDa的低分子量蛋白质（LMW）的研究（Torabi等人，2014）。
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