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TWEEN® 20

TWEEN ® 20

Company: Sigma-Aldrich
Catalog#: P1379
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Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
Author:
Date:
2018-06-20
[Abstract]  Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular ... [摘要]  双链RNA是一种有效的病原体相关分子模式(PAMP),作为病毒复制的副产物和病毒感染的众所周知的标志产生。 病毒dsRNA可以从感染的细胞释放到细胞外空间并通过胞吞作用被邻近细胞内化。 哺乳动物具有能够检测导致产生I型干扰素(IFN)的病毒dsRNA(例如内体Toll样受体3(TLR3)和胞质RIG-1样受体(RLR))的多模式识别受体(PRR)。 因此,病毒dsRNA的细胞内定位可以提供对导致先天免疫激活的下游信号传导途径的了解。 在这里,我们描述了一种测量细胞外dsRNA摄取和分别通过流式细胞仪和共聚焦显微镜观察内化dsRNA的亚细胞定位的定量方法。

【背景】双链RNA(dsRNA)是病毒复制的常见副产物,通过产生I型干扰素(IFN)和其他促炎细胞因子(Nellimarla和Mossman,2014)是抗病毒免疫的有效激活剂。病毒的dsRNA通过TLR3核内体内所感测(松本等人,2003年)或在由RIG-I样受体(RLRS),RIG-I和MDA-5(加藤等人,2006)。在裂解感染,这些dsRNA可以被释放到细胞外空间,它们结合于相邻小区,如A类清道夫受体(SR-A)和RAFTLIN表面受体,并且随后经由网格蛋白介导的内吞作用内在化(伊藤制 2008; DeWitte-Orr等人,2010; Watanabe等人,2011; Dansako等人,, ...

Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings
Author:
Date:
2018-06-05
[Abstract]  Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation – one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). [摘要]  非洲特有的稻黄斑驳病毒(RYMV)是一种机械传播的病毒,对栽培稻株造成严重损害。 品种抗性选择被认为是最有效和可持续的管理策略。 需要标准化的阻力评估程序来识别和表征阻力源。 本文介绍了利用RYMV机械接种水稻幼苗的方案,以及两种抗性评估方法 - 一种基于症状严重性指数,另一种基于双抗体夹心酶联免疫吸附试验(DAS-ELISA)进行病毒检测。

【背景】RYMV是非洲水稻生产的主要生物限制因素(Séréet al。,2013),在非洲和马达加斯加的大多数水稻生产国都有报道。它不能通过种子传播(Konaté等人,2001; Allarangaye等人,2006),而是通过昆虫载体(特别是甲虫)和农业作业期间的接触传播Bakker,1974;Traoré等人,2006),尤其是在将苗床的苗移植到田间时。该病毒非常稳定,能够在大米和野生禾本科(Bakker,1974)宿主中以高浓度繁殖。

监测苗床RYMV发病率和品种选择是管理RYMV最有效和可持续的方式。存在两种表型的抗性 - 部分抗性,其特征在于症状出现延迟(Albar等人,1998)和高抗性,其特征在于使用DAS-ELISA不存在病毒检测(Ndjiondjop et al。,1999)。虽然部分抗性在水稻品种中广泛分布,但栽培水稻品种仅有少数品种O.水稻和 O。 glaberrima ...

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
Author:
Date:
2018-01-05
[Abstract]  This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and ... [摘要]  该协议通过下拉共免疫沉淀分析两种DNA结合蛋白之间的直接相互作用。其中一种蛋白在E中过表达。如HA标记的重组蛋白和无细胞提取物在HA亲和树脂中免疫沉淀。用核酸酶处理细胞提取物以降解DNA和RNA,这排除了核酸介导的间接相互作用。然后,使用纯化的推定的配偶体蛋白进行第二次免疫沉淀步骤。如果特异性抗体可用,可以通过考马斯蓝染色和/或Western印迹(WB)检测免疫共沉淀蛋白质。此外,许多DNA / RNA结合蛋白具有高度正电性,在标准条件下可阻碍WB,正如组蛋白和组蛋白样蛋白所示。在这种情况下,我们表明,假定的合作伙伴的高等电点导致转移不良。提示麻烦WB提供高正电荷DNA结合蛋白的转移。


【背景】共免疫沉淀是分析蛋白质 - 蛋白质相互作用(PPI)的常用方法。许多共免疫沉淀方案使用细菌表达的蛋白质。然而,细胞提取物的使用不排除由第三种蛋白介导的间接相互作用,或者在DNA / RNA结合蛋白的情况下介导核酸。

乙型病毒Bam35(B35TP)的末端蛋白含有保守的酪氨酸194,其提供OH基团以在蛋白质引发的DNA复制期间锚定病毒基因组的第一个5'-dTMP。此外,B35TP具有很强的DNA结合能力,与许多DNA结合蛋白一样,它具有非常高的等电点(约10.6),这影响其体外稳定性和功能(Berjón-Otero 等),2016)。 ...

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