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E-64

E-64

Company: Sigma-Aldrich
Catalog#: E3132
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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。

Protein Sample Preparation for Proteomic Analysis in Leishmania donovani
Author:
Date:
2014-03-05
[Abstract]  Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional over pre-transcriptional regulatory mechanisms (Clayton, 2002). Thus proteomic analysis has proven an essential tool for understanding pathways implicated in Leishmania infectivity, host-parasite interactions, drug resistance and others. When employing a comparative proteomics analysis between different parasitic cell lines, it is essential that these lines are cultivated in exactly the same way, in the same cell density and growth phase. More importantly when ... [摘要]  利什曼原虫属是锥虫病原生动物属,是负责疾病利什曼病的寄生虫。这些原生动物,与其他高等真核生物相比,以非典型方式调节它们的基因表达。基因表达的调节的特征在于转录后优先于转录前调节机制(Clayton,2002)。因此,蛋白质组学分析已经证明是了解利什曼原虫感染,宿主寄生虫相互作用,耐药性和其他相关途径的重要工具。当在不同寄生细胞系之间使用比较蛋白质组学分析时,以相同的方式,在相同的细胞密度和生长期中培养这些细胞系是必要的。更重要的是,当怀疑细胞周期缺陷时,必须在同一细胞周期阶段同步细胞系,以消除可能的人工因素。该方案描述了在杜氏利什曼原虫( donovani )中用于蛋白质组分析的全蛋白样品的制备。

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