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Amersham ECL Western Blotting Detection Reagent

Amersham ECL Western Blotting Detection Reagent

Company: GE Healthcare
Catalog#: RPN2106
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In vitro Enzymatic Assays of Histone Decrotonylation on Recombinant Histones
Author:
Date:
2018-07-20
[Abstract]  Class I histone deacetylases (HDACs) are efficient histone decrotonylases, broadening the enzymatic spectrum of these important (epi-)genome regulators and drug targets. Here, we describe an in vitro approach to assaying class I HDACs with different acyl-histone substrates, including crotonylated histones and expand this to examine the effect of inhibitors and estimate kinetic constants. [摘要]  I类组蛋白去乙酰化酶(HDACs)是有效的组蛋白去蛋白酶,拓宽了这些重要(epi-)基因组调节因子和药物靶标的酶谱。 在这里,我们描述了一种体外方法来测定具有不同酰基 - 组蛋白底物的I类HDAC,包括巴豆酰化组蛋白,并将其扩展以检查抑制剂的作用并估计动力学常数。

【背景】组蛋白的翻译后修饰是基因组调控的重要方面,包括基因表达(例如参见Pengelly et al。,2013;在Castillo 等人中综述,2017)。组蛋白修饰改变染色质结构和/或调节蛋白质的结合,例如核小体重塑因子(在Bannister和Kouzarides,2011中综述)。大多数组蛋白修饰是可逆的并且可以酶促去除。例如,通过组蛋白脱乙酰基酶(HDAC)除去组蛋白乙酰化,其中存在几类。近年来,新的组蛋白赖氨酸酰化,包括琥珀酰化,丙酰化,丁酰化,羟基丁基化和巴豆酰化已成为规范组蛋白乙酰化的新替代物,并且已经证实了许多这些新发现的组蛋白修饰的功能相关性(Sabari 等人,2017)。特别是,组蛋白巴豆酰化与活性基因表达有关,并被认为受细胞代谢状态的影响(Sabari et al。,2015; Fellows et al。 ,2018年)。最近已显示I类组蛋白脱乙酰酶也有效地去除组蛋白质(Wei et al。,2017; Fellows et al。,2018)。
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An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
Author:
Date:
2017-11-20
[Abstract]  We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et ... [摘要]  我们最近报道了一种针对内源性感兴趣蛋白(POI)的靶向蛋白水解的亲和指导PROtein导弹(AdPROM)系统(Fulcher等人,2016和2017)。 AdPROM由Von Hippel Lindau(VHL)蛋白组成,Cullin 2 E3连接酶底物受体(Bosu and Kipreos,2008),与识别细胞中靶蛋白的高亲和力多肽结合剂(如骆驼科纳米抗体)缀合。当在细胞中引入时,靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集,我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体(Sharm等人,2013; Fridy等人。,2014; Schmidt et al。,2016)。在此协议中,我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征,并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性,这些方法需要长时间的治疗并与脱靶效应相关联,而CRISPR / Cas9基因编辑并不总是可行的。
【背景】该协议使人们能够在哺乳动物细胞系中设计,构建和表达AdPROM VHL-nano ...

Detection of ASC Oligomerization by Western Blotting
Author:
Date:
2017-05-20
[Abstract]  The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot. [摘要]  具有半胱天冬酶募集区(ASC)衔接蛋白的凋亡相关斑点样蛋白桥联炎症体传感器和半胱天冬酶-1。在炎性体活化后,ASC以类似朊病毒的方式成核,成为负责募集和激活半胱天冬酶-1的大而单一的平台。活性胱天蛋白酶-1将反过来促进促炎细胞因子IL-1β的蛋白水解成熟。 ASC寡聚化是炎性体激活的直接证据,其检测允许读取与caspase-1和IL-1β无关。该方案描述了如何通过蛋白质印迹检测ASC的寡聚化。

背景 Inflammasomes是大量的多蛋白平台,其感测各种微生物,内源和环境胁迫因子,导致促炎IL-1细胞因子家族的成熟(Martinon等人,2002; Sharma和Kanneganti, 2016)。激活后,炎性细胞传感器通过pyrin结构域(PYD)-PYD同型相互作用募集衔接蛋白ASC。 ASC通过胱天蛋白酶激活和募集域(CARD)-CARD相互作用又结合半胱天冬酶-1,并有利于caspase-1的自我蛋白水解切割,导致IL-1β和IL-18的成熟(Hoss等人。,2016)。 Inflammasome激活引发ASC二聚体的超分子寡聚化成称为“ASC-specks”或“pyroptosome”(Fernandes-Alnemri等人,2007)的大交织原纤维。 ASC-speck / ...

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