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HiPrepTM 16/60 Sephacryl® S-300 HR

色谱柱

Company: Cytiva
Catalog#: 17-1167-01
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Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
Author:
Date:
2017-04-05
[Abstract]  Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized by recipient neighboring or distal cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. Exosomes facilitate intercellular communication, modulate cellular phenotype, and regulate microbial pathogenesis. We have previously shown that semen exosomes (SE) inhibit HIV-1 replication in various cell types. Here, we describe detailed ... [摘要]  外来体是内膜起源的膜性胞外纳米囊。 已知外来载体携带宿主和病原体衍生的基因组,蛋白质组,脂质体载体和其他外来分子。 外来体由不同细胞类型分泌到细胞外环境中,随后被受体相邻细胞或远端细胞内化。 在内化后,外来体通过捐赠其载体和或激活各种信号转导途径来调节受体细胞,从而调节生理和病理生理过程。 外来体促进细胞间通讯,调节细胞表型和调节微生物发病机制。 我们以前表明精液外来体(SE)抑制各种细胞类型的HIV-1复制。 在这里,我们描述特征SE的详细协议。 该方案可以适应或修改,并用于评估感兴趣的其他细胞外小泡。

外来体是由许多细胞类型的晚期内体室内的内体膜向内发生的结果而引起的膜状纳米囊(Simons and Raposo,2009)。外来体被许多细胞类型(Iglesias等人,2012)释放到细胞外环境中,并且被发现在包括血液在内的生物流体中(Kaur等人,2014)尿(Liem等人,2013)唾液(Madison等人,2015)和母乳(Madison等人,2014; Naslund ,2014)。人类精液含有由包括前列腺分泌腺泡细胞在内的男性生殖道组织产生的纳米囊泡的异质群体(Madison等人,2014; Madison等人,2015) (Sahlen等人,2002)和附睾上皮细胞(Frenette等人,2010)以及vasa感染,睾丸和囊泡腺细胞( ...

In vitro Deneddylation Assay
Author:
Date:
2016-03-20
[Abstract]  Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western ... [摘要]  Nedd8是共价连接到作为cullin-RING连接酶(CRL)的一部分的cullin蛋白的保守赖氨酸残基的小的遍在蛋白样蛋白(9kDa)。 CRL是对泛素 - 蛋白酶体途径(UPP)中的蛋白质泛素化重要的主要E3连接酶。 CRL的活性通过脱甲基化(CulA-N8,〜98kDa)和脱甲基化(CulA〜89kDa)的循环调节。 COP9信号体(CSN)和丁二酸酶A(DenA)能够切割Nedd8和CullinA之间的异肽键。与单一蛋白DenA相反,CSN是八亚基多蛋白复合物。将不同的构巢曲霉csn 缺失菌株的蛋白质粗提物与从大肠杆菌(大肠杆菌)表达和纯化的重组CSN亚基混合。使用抗CulA或抗Nedd8抗体的Western杂交实验可以显示Nedd化化合物与Denedd化CulA的比率。使用deneddylation测定,我们可以显示CsnE是在体外连接7-亚基预组装的CSN的最后一个亚基,然后CSN可以通过金属蛋白酶亚基CsnE执行cullin deneddylation。该测定法是一种快速且非昂贵的方法,其显现了用于脱蛋白的蛋白质的酶活性。它还可用于测试除去来自构巢曲霉(构巢曲霉)或其他生物体中的底物的翻译后修饰的其它藻肽的活性。

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