| Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana
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Author:
Date:
2018-06-20
[Abstract] As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.
A number of regulators have been characterized in plants, including the autophagy-related (ATG) proteins and other plant-specific proteins. Among them, ATG8 will undergo a lipidation process to become a membrane-bound ATG8-phosphatidylethanolamine form and mark the growing autophagosomal membrane as well as the completed autophagosome. Therefore, ATG8 has been regarded as a marker for autophagosomes; and biochemical detection of the membrane-associated form of ATG8 is used as one of ...
[摘要] 作为降解和回收细胞货物的基本代谢途径，自噬在植物的胁迫，饥饿和衰老条件下被高度诱导。 在这个过程中，将形成一个称为自噬体的双膜结构，用于货物隔离和输送到液泡中。
已经在植物中表征了许多调控因子，包括自噬相关（ATG）蛋白和其他植物特异性蛋白。 其中，ATG8将经历脂化过程以成为膜结合的ATG8-磷脂酰乙醇胺形式并标记日益增长的自噬体膜以及完成的自噬体。 因此，ATG8被认为是自噬体的标志; 并且膜结合形式的ATG8的生物化学检测被用作测量自噬活性的主要方法之一。 在这里，我们描述了使用拟南芥幼苗检测ATG8-PE形式的ATG8脂化测定法。
【背景】自噬是调节受损细胞器的大量降解和不需要的细胞内容物的基本代谢过程。在自噬过程中，称为自噬体的双膜结构将形成并将货物递送到液泡中以降解。自噬相关蛋白（ATG）需要调节自噬活性（Liu and ...
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| Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
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Author:
Date:
2017-04-20
[Abstract] Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.
This ...
[摘要] 尺寸排阻色谱法（SEC）或凝胶过滤是一种流体动力学技术，它将溶液中的分子作为其尺寸和形状的函数。在蛋白质的情况下，可以通过实验得出的流体动力学值是斯托克斯半径（R s），它是具有相同流体力学性质的球体的半径（即， >摩擦系数）作为生物分子。通过SEC测定R s已经被广泛用于监测由钙（Ca 2+）与许多Ca 2+连接引起的构象变化传感器蛋白。对于这类蛋白质，SEC分离不仅基于Ca 2 + 结合后的蛋白质尺寸变化，而且可能来自水合壳结构的变化。
该方案旨在使用快速蛋白质液相色谱（FPLC）系统对预填充柱进行凝胶过滤实验，以确定蛋白质的R 1，其中一些适用于Ca 2 + 传感器蛋白。
凝胶过滤基于其相对的能力分离不同大小和形状的分子，以穿透具有明确孔径的多孔珠床，其识别分馏范围。大于完全排除进入孔隙的分馏范围的分子快速流过色谱柱，首先以空间体积（V 0 O）（其为支持颗粒外的间隙体积）洗脱。能够扩散到珠的孔中的小于分级范围的分子具有可用于流动相的总体积，因此它们更缓慢地移动通过床并最后洗脱。具有中等维度的分子将以包含在流动相可利用的空隙体积和总体积之间的洗脱体积（V e e e e）被洗脱（分子越小，其进入孔隙越大矩阵，因此其V e e越大）。
蛋白质的分子量可以通过比较其洗脱体积参数K ...
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| The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants
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Author:
Date:
2015-12-05
[Abstract] Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70, this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were ...
[摘要] Pepino花叶病毒（ PepMV ）是一种机械传播的病原体，影响全世界的番茄植株。与其他植物病毒一样（Verchot，2012），热休克同源蛋白70同源物（Hsc70）被鉴定为PepMV外壳蛋白（CP）的相互作用物（Mathioudakis等人，2012）。在这里，我们描述了使用槲皮素使沉默Hsp70在植物中的药理学方法（Mathioudakis等人，2014），Hsp70蛋白质表达类黄酮抑制剂（Hosokawa等人，1990 ; Manwell和Heikkila 2007）。在Hsp70的情况下，该方法表示比通过反向遗传学测定（例如VIGS方法）沉默Hsp70更快和更容易的方法。使用注射器用槲皮素（溶解于DMSO中）或DMSO（对照植物）浸润2至3周龄的烟草烟草的完全膨胀的叶子。用PepMV病毒接种物机械接种植物。在接种后4天通过免疫印迹分析在局部叶子上分析Hsp70和PepMV的积累。
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