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Sodium dodecyl sulfate (SDS)

Company: Merck
Catalog#: L3771
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In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
Author:
Date:
2021-04-05
[Abstract]  

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously

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[摘要]  [摘要]大多数细胞蛋白的s的降解通过26S在真核生物蛋白酶。但是,内在无序的蛋白质(IDPs)包含大量的非结构化区域,并且内在地不稳定,因此很容易通过不依赖泛素的20S蛋白酶体降解。越来越多的证据最近显示ň植物境内流离失所者的平衡也可以通过20S蛋白酶控制。但是,由于缺乏用于体外分离20S蛋白酶体和降解测定的系统协议,因此我们对植物中IDP和20S蛋白酶体降解的功能和调控机制的研究和理解一直处于婴儿期与其他生物。在这里,我们通过采用和修改先前公开的方法,对拟南芥中20S蛋白酶体进行体外重组测定的详细方案。在此获得20S核心蛋白酶体的主要策略是从26S蛋白酶体中去除19S调节亚基。该协议包括两个主要部分:1)的来自表达表位标记的PAG1稳定的转基因品系20S蛋白酶体亲和纯化,的20S蛋白酶(程序AD)的基本组成部分; 2 )体外20S蛋白酶体降解测定法(方法E)。我们预计该协议将提供一种简单有效的方法来研究体外20S蛋白酶体降解,并促进植物中蛋白质代谢的研究。

[背景]蛋白质的降解通常是通过真核生物中的蛋白酶体来实现的。整合的26S蛋白酶体由两个亚颗粒组成:一个或两个末端的19S调节颗粒(RP),用作蛋白酶体激活剂;和20S核心蛋白酶体(CP),执行降解过程。大多数真核蛋白被多聚泛素化并导入26S蛋白酶体进行降解。然而,含有固有蛋白质无序已发现的区域直接通过破坏一个由20S蛋白酶的泛素依赖性降解(本日产等人,2014) ...

EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
Author:
Date:
2021-02-20
[Abstract]  

Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer’s. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be

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[摘要]  [摘要]转录错误可通过改变表观基因组并引起mRNA的错误整合而严重影响生物体内的代谢过程,从而将其翻译为异常的突变蛋白。此外,真核基因组内有特定转录错误富集的基因组基因座(TEELs),它们由RNA聚合酶与显著更高的错误率转录并推测为具有影响在癌症,老化和疾病例如唐氏综合征和阿尔茨海默'秒。因此,在遗传学领域对转录错误的研究越来越重要。尽管如此,方法上的障碍限制了准确识别转录错误的进展。Pro-Seq和NET-Seq可以沿基因组纯化新生RNA并绘制RNA聚合酶,但不能用于鉴定转录突变。在这里,我们本背景误差模型耦合的精密核圆形测序上运行(EMPC -SEQ),一种方法COMBIN荷兰国际集团测定和圆形测序核上运行与背景误差模型精确地检测新生转录错误和有效地辨别TEELs基因组中。

[背景]核糖核苷酸错掺导致的转录错误在所有活生物体中无处不在(Carey,2015)。假设每个信使RNA(mRNA)可以翻译2-4千次(Schwanhausser et al。,2011),并且许多特殊RNA在给定时间每个细胞仅表达一次(Islam et al。,2011; Pelechano et al。,2011)。,2010),即使是关键残基的单个转录错误也会使特定蛋白质的表达产生很大差异。另外,转录错误可加速蛋白质聚集,导致人类中与年龄有关的疾病(van ...

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