| Cycloheximide Assays to Measure Protein Degradation in vivo in Plants
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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。
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| Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues
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Author:
Date:
2016-07-20
[Abstract] Fusarium graminearum (Fg) is the causal agent of Fusarium head blight disease of wheat (Triticum aestivum), oats (Avena sativa) and barley (Hordeum vulgare), which targets the floral tissues and thereby adversely impacts grain yield and quality. Mycotoxins produced by F. graminearum further limit the consumability of infected grain. In the laboratory, F. graminearum also has the ability to colonize both leaves and inflorescence tissues of Arabidopsis thaliana. The interaction between A. thaliana and F. graminearum makes available a large array of genetic and molecular tools to study the interaction between plants and F. graminearum to elucidate plant genes and pathways that contribute to resistance, ...
[摘要] 禾谷镰刀菌（Fg）是小麦（小麦），燕麦（ Avena sativa ）和大麦镰刀菌 ），其针对花组织，从而不利地影响谷物产量和质量。由F生产的霉菌毒素。禾谷镰菌进一步限制了感染谷物的消耗性。在实验室，F。禾谷镰刀菌也具有定居拟南芥的叶和花序组织的能力。 A之间的交互。 thaliana 和 F。禾谷镰刀菌（graminearum）提供了大量遗传和分子工具来研究植物和真菌之间的相互作用。禾本科（Graminearum）来阐明植物基因和促进抗性的途径，以及研究真菌如何靶向植物基因和促进疾病的机制。下面描述的方法允许有效地感染拟南芥叶和花序，以及评价疾病进展和真菌生长。拟南芥中的病害传播可以通过叶组织的萎黄病和花序组织的包括真菌团块在花序组织表面上的病害表型的视觉观察来容易地监测。可以通过聚合酶链反应（PCR）和定量实时PCR（qPCR）测量宿主组织中的Fg DNA的相对量来进一步监测真菌生长。
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| Aβ Extraction from Murine Brain Homogenates
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Author:
Date:
2016-04-20
[Abstract] This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals.
[摘要] 该方案详述了从转基因鼠脑匀浆中提取β-淀粉样蛋白（Aβ）。 具体来说，详细说明脑组织的机械均质化和可溶性和不溶性蛋白质的顺序提取。 DEA提取可溶性蛋白质，如Aβ异构体和APP。 甲酸能够提取不溶性蛋白质聚集体，例如与斑块相关的Aβ同种型。 该方法产生可溶性和不溶性提取物，其适于通过western印迹和/或酶联免疫吸附测定（ELISA）分析Aβ物质，并且这些结果有助于评估动物的淀粉样变性负担。
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