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Tricine

Tricine

Company: Sigma-Aldrich
Catalog#: T0377
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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。

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp
Author:
Date:
2015-12-20
[Abstract]  This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development. [摘要]  这个协议描述了从番茄果实隔离tonoplast囊泡。 使用该程序分离的囊泡具有足够高的纯度用于下游蛋白质组分析,同时保留用于功能测定的转运感受态。 该方法用于研究番茄果实成熟期间氨基酸的转运(Snowden等人,2015)并且基于Betty和Smith使用的程序(Bettey和Smith,1993)。 这样的囊泡可用于进一步研究代谢物在整个质膜上的动态转移,用于番茄果实发育期间的储存和代谢。

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