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The Gradient Master

渐变主人

Company: USA Scientific
Catalog#: 107-201M
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Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
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Date:
2017-04-20
[Abstract]  The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016). [摘要]  RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在,外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用(参见[Kilchert等人,2016])。以下方案描述了从HEK-293细胞中纯化RNA外来体复合物的方法,利用可诱导的异位表达,亲和力捕获和速率 - 区带离心。所获得的RNA外来体已被成功地用于蛋白质组学,结构和酶学研究(Domanski等人,2016)。

在我们以前的工作中,我们建立了在四环素诱导型CMV启动子(HEK-293 Flp-In T-REx-Thermo Fisher)的控制下表达C末端3xFLAG标记的外来体组分EXOSC10(RRP6)的同基因HEK-293细胞系科学)。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质,并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案(Domanski等, / em>。,2012)。进一步探索使用的蛋白质提取条件,我们开发了一种允许在亲和捕获的外来体内保留DIS3(RRP44)的方案,否则证明是困难的(Hakhverdyan等人,2015)。基于这些研究,我们通过使用甘油密度梯度的速率区带离心法进一步纯化了RNA外来体(+/- DIS3)(Domanski等人,2016)。虽然蛋白质提取物中洗涤剂CHAPS(3 - ...

Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
Author:
Date:
2016-05-20
[Abstract]  Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). ... [摘要]  呼吸道合胞病毒(RSV)属于包括许多临床相关病毒(例如人偏肺病毒和麻疹)的副粘病毒家族。 RSV感染可以在婴儿,老年人和一些免疫受损的成人中引起严重的疾病。在RSV复制期间,产生一系列截短形式的病毒基因组。这些截短的病毒基因组被称为缺陷型病毒基因组(DVG),并且由许多病毒产生(Lazzarini等人,1981; Rao和Huang,1982; Prince等人, ,1996; Sun等人,2015; Tapia等人,2013)。 DVG可以限制全长病毒的复制,并且是针对RSV的先天免疫应答的主要天然触发因子(Sun等人,2015; Tapia等人 ,2013)。在这里,我们详细讨论如何准备具有高或低含量的DVG的RSV股票,以及如何从富含缺陷病毒颗粒的RSV原液中纯化含有DVG的缺陷病毒颗粒。这些程序可用于制备实验室研究所需的病毒原液和有缺陷的病毒颗粒。简言之,在HEp2细胞中产生不同的RSV种群,其通常用于在实验室中扩增该病毒。为了产生具有高含量DVG的RSV原液,HEp2细胞用高多重感染(MOI)多次感染,然后使用梯度离心纯化含有DVG的病毒颗粒。这里描述的方法具有四个部分:1.具有低DVG含量(RSV-LD)的种子RSV原种的扩增,2.具有高DVG含量(RSV-HD)的原种的产生,3.通过梯度离心,4.纯化DVG的表征。

Purification of 70S Ribosomes from Bacillus subtilis
Author:
Date:
2015-04-05
[Abstract]  The eubacterial ribosome (70S) is a macromolecular complex that is composed of a small (30S) subunit and a large (50S) subunit. The small subunit comprises the 16S ribosomal RNA (rRNA) and more than 20 ribosomal proteins (r-proteins), whereas the large subunit comprises the 23S and 5S rRNAs and more than 30 r-proteins. Bacillus subtilis (B. subtilis) has 57 r-ribosomal protein genes and three rRNAs (16S, 23S and 5S rRNAs). Among them, we identified 21 r-proteins of the small subunit and 31 r-proteins of the large subunit in B. subtilis (Nanamiya et al., 2004). The functions and roles of individual components of the ribosome have not yet been completely clarified. Herein we describe in detail an ultracentrifugation-based protocol for the preparation of ... [摘要]  真细菌核糖体(70S)是由小(30S)亚基和大(50S)亚基组成的大分子复合物。 小亚基包含16S核糖体RNA(rRNA)和超过20个核糖体蛋白(r-蛋白),而大亚基包含23S和5S rRNA和超过30个r蛋白。 枯草芽孢杆菌(<枯草芽孢杆菌)具有57个核糖体蛋白基因和三个rrna(16s,23s和5s rrna)。 其中,我们确定21 r蛋白的小亚基和31 r蛋白的大亚基在b。 枯草芽孢杆菌(nanamiya等人,2004)。 核糖体的各个组分的功能和作用尚未完全澄清。 在本文中,我们详细描述了从b的指数生长细胞制备70s核糖体的基于超速离心的方案。 枯草芽孢杆菌。 rrna)。="" 其中,我们确定21="" r蛋白的小亚基和31="" r蛋白的大亚基在b。="" 枯草芽孢杆菌(nanamiya等人,2004)。="" 核糖体的各个组分的功能和作用尚未完全澄清。="" 在本文中,我们详细描述了从b的指数生长细胞制备70s核糖体的基于超速离心的方案。="" 枯草芽孢杆菌。=""> ...

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