| Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
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Author:
Date:
2016-08-20
[Abstract] VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to ...
[摘要] VPS34是哺乳动物细胞中唯一的III类磷脂酰肌醇-3-激酶（PI3K），并且产生绝大多数细胞磷脂酰肌醇-3-磷酸[PI（3）P]。 PI（3）P是一个关键的信号脂质，在诸如内吞作用和自噬的过程中起许多膜运输的作用。 VPS34是关键的细胞调节剂，功能丧失可具有灾难性作用并且是胚胎致死的（Zhou等人，2011）。 细胞PI（3）P的水平可以通过荧光染色技术确定，并且可以用于监测对VPS34活性的影响，然而重要的是验证任何变化由VPS34介导，特别是作为PI（3）的替代途径， P生产是可能的，例如通过II类PI3K（Devereaux等人，2013）。 直接在体外测定VPS34活性可以是描绘特定刺激的作用的关键阶段。
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| Purification of Bacterial RNA from Infected Macrophages
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Author:
Date:
2015-11-20
[Abstract] Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to ...
[摘要] 在感染期间研究细菌病原体的转录组是一个非常有益的和有效的工具，用于发现有助于成功感染的基因。然而，从感染的细胞或组织中分离细菌RNA是一个挑战性的过程，因为感染细胞裂解物中宿主RNA的量高得多。我们已经优化了用于分离感染骨髓来源的巨噬细胞（BMDM）的单核细胞增生性李斯特菌（（单核细胞增生李斯特氏菌）细菌）的RNA的方法。感染后，我们裂解细胞并通过0.45μm过滤器过滤裂解物以丢弃大多数宿主蛋白和RNA。接下来，我们重新悬浮细菌，并在DNase处理后提取RNA。提取的RNA适合于通过实时PCR或微阵列的基因表达分析。我们已经在我们对感染期间的单核细胞增生李斯特氏菌基因调节的体外研究中成功地采用了该方案（Lobel等人，2015; Lobel >，2012; Kaplan Zeevi等人，2013; Rabinovich等人，2012）。
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| Extraction of Total Proteins from Rice Plant
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Author:
Date:
2014-11-05
[Abstract] This protocol provides an efficient method for preparation of high-quality proteins from rice leaves and grains. The method involves phenol extraction to separate proteins from the non-protein components such as polysaccharides, lipids and phenolic compounds that are commonly enriched in plant tissues. Following isolation, proteins are precipitated with ammonium acetate/methanol and then solubilized for proteomic analysis. As the protocol is simple, universal, and most importantly compatible with silver staining, it has been applied to our routine protein extraction from rice and many other ...
[摘要] 这个协议提供了一种从稻叶和谷物制备高质量蛋白质的有效方法。 该方法包括酚提取以从通常富含植物组织的非蛋白质组分如多糖，脂质和酚类化合物中分离蛋白质。 分离后，用乙酸铵/甲醇沉淀蛋白质，然后溶解用于蛋白质组分析。 由于协议简单，通用，最重要的是与银染色兼容，它已经应用于我们从水稻和许多其他植物组织的常规蛋白质提取，甚至在动物组织中对于电泳分离的要求很好。
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