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Mini-PROTEAN® Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel

Mini-PROTEAN ® Tetra Vertical Electrophoresis Cell for Mini Precast Gels,4-gel

Company: Bio-Rad Laboratories
Catalog#: 1658004
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BMV Propagation, Extraction and Purification Using Chromatographic Methods
Author:
Date:
2018-07-20
[Abstract]  Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral capsids in nanotechnology. Herein, we describe a method for BMV purification based on ion-exchange and size-exclusion chromatography. The presented method is of similar efficiency to previously described protocols relying on differential centrifugation and can easily be scaled up. The resulting BMV capsids are stable and monodisperse and can be used for further applications. [摘要]  雀麦花叶病毒(BMV)是众所周知的植物病毒,代表单链RNA(ssRNA)正义病毒。 它已被广泛用作植物病毒生物学,流行病学和病毒衣壳在纳米技术中的应用的多项研究中的模型。 在本文中,我们描述了基于离子交换和尺寸排阻色谱的BMV纯化方法。 所提出的方法与先前描述的依赖于差速离心的方案具有相似的效率,并且可以容易地按比例放大。 得到的BMV衣壳是稳定的并且是单分散的,并且可以用于进一步的应用。

【背景】纳米技术要克服的关键挑战之一是制定有效的和组织特异性的药物递送方法。植物病毒和病毒样颗粒(VLP)具有生物相容性和可生物降解性,不含对人类或动物健康有害的病原体,是合成药物载体的安全替代品,通常会激活免疫系统的不良反应或积聚在免疫系统中。身体到毒性水平。最后,病毒衣壳的生产相对便宜且快速(Ren et al。,2007; Arcangeli et al。,2014)。

Bromoviridae 家族的雀麦花叶病毒(BMV)是用作纳米颗粒载体的良好候选物,因为它显示出所有上述特征并且是研究最多的植物病毒之一(Figlerowicz,2000; Alejska et al。,2005; Urbanowicz et al。,2005; Wierzchoslawski et al。,2006; Kao vet al。 ...

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)。 ...

Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
Author:
Date:
2017-08-05
[Abstract]  Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of ... [摘要]  集群定期间隔短回归重复(CRISPR)和CRISPR相关(Cas)系统作为细菌和古菌中的适应性免疫系统,用于防御入侵病毒和质粒(Barrangou和Marraffini,2014)。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向(Jinek et al。,2012; Cong等人,2013; Mali ,2013; Zetsche等人,2015)。然而,真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9(SpCas9)靶向DNA接近的障碍,并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ (Knight等人,,2015; Hinz等人,2015; Horlbeck等人,2016年) ; ...

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