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Tricine

Tricine

Company: Sigma-Aldrich
Catalog#: T0377
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Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
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。 ...

Cycloheximide Assays to Measure Protein Degradation in vivo in Plants
Author:
Date:
2016-09-05
[Abstract]  The half-life of a protein is a characteristic property, and can be modulated by post-translational modifications, changes in subcellular localization, and/or interaction with other proteins or ligands. As one determinant of its steady-state level, a protein’s degradation represents an important distinguishing attribute relevant to its biological function. Because protein longevity cannot be elucidated from bioinformatics analyses, it must be determined empirically. Here we describe two approaches for in vivo half-life determination in plants: 1. pooled-seedling degradation assays monitoring either tagged versions of the protein (luciferase fusions or other epitope tags) or following the endogenous protein; 2. single-seedling degradation assays using luciferase fusion proteins. ... [摘要]  蛋白质的半衰期是特征性质,并且可以通过翻译后修饰,亚细胞定位的改变和/或与其他蛋白质或配体的相互作用来调节。 作为其稳态水平的一个决定因素,蛋白质的降解代表与其生物学功能相关的重要的区别属性。 由于蛋白质的寿命不能从生物信息学分析中阐明,必须根据经验确定。 这里我们描述了在植物中体内半衰期测定的两种方法:1.监测蛋白质(萤光素酶融合物或其它表位标签)的标记形式或遵循内源蛋白质的混合 - 幼苗降解测定; 2.使用荧光素酶融合蛋白的单胚芽降解测定。 这些方法的优点是它们的简单性和低成本。

Subchromoplast Fractionation Protocol for Different Solanaceae Fruit Species
Author:
Date:
2016-07-05
[Abstract]  Macromolecules, proteins, lipids, and other small molecules, such as carotenoids can be studied within different tissues and organelles using an array of in vitro and in vivo methodologies. In the case of tomato and other fleshy fruit the predominant organelle in ripe fruit is the chromoplast. The characteristic feature of this organelle is the presence of pigments, carotenoids at high levels. In order to fully understand the underlying biological mechanisms that operate within the chromoplast, it is necessary to perform studies at the subchromoplast level. This protocol allows the separation of plastoglobules (lipoprotein particles, which are coupled to thylakoid membranes in the chloroplasts) and membranes (thylakoid, envelope-like) of chromoplasts through a sucrose ... [摘要]  可以使用体外和体内方法的阵列在不同的组织和细胞器中研究大分子,蛋白质,脂质和其它小分子,例如类胡萝卜素。在番茄和其他肉果的情况下,成熟果实中的主要细胞器是色质体。这种细胞器的特征是存在高水平的颜料,类胡萝卜素。为了完全理解在chromoplast内操作的基本生物机制,有必要在subchromoplast水平进行研究。该方案允许通过蔗糖梯度分离塑料球(脂蛋白颗粒,其与叶绿体中的类囊体膜偶联)和膜(类囊体,包膜样)的色原体。然后可以独立地分析亚氯化银色室。突变体/转基因基因型与其背景之间的比较可以在同一分馏运行期间同时进行精确进行。该程序最初是为了成熟番茄果实开发的,但已经显示翻译成甜和辣椒。

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