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Ammonium peroxydisulphate

过二硫酸铵

Company: Carl Roth
Catalog#: 9592
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Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
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Date:
2018-10-05
[Abstract]  Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ... [摘要]  核糖核蛋白颗粒(mRNP)是由mRNA和RNA结合蛋白(RBP)组成的复合物,其控制mRNA转录定位,转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件,如poly(A)尾或5'帽结构,这通过应用广泛使用的方法如oligo(dT)纯化来阻止它们的鉴定。在这里,我们描述了基于现有的交联和免疫沉淀(CLIP)方法的修饰的交联亲和纯化方案(cCLAP),以分离mRNP中可被去腺苷酸化,去除和/或部分降解的mRNA,从而开启了检测不同的可能性。非编码RNA(ncRNA)的类型。分离后,将RNA进行衔接子连接,然后进行下一代测序(NGS)。由于快速有效的交联和淬灭步骤,该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体(PB)或应力颗粒(SG)。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀,然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法(Tagwerker et al。,2006; Hafner et al。,2010; Kishore et al。,2011)。 ...

ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors
Author:
Date:
2017-04-05
[Abstract]  The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., ... [摘要]  肌动蛋白相关蛋白2/3(ARP2 / 3)复合物是在哺乳动物细胞中产生支链肌动蛋白网络的肌动蛋白成核剂。除了结合成核促进因子之外,LeClaire等人。证明其磷酸化状态是其活性的关键(LeClaire等人,2008)。在细胞中,ARP2 / 3复合物在ARP2,ARP3和ARPC1亚基的苏氨酸和酪氨酸残基上磷酸化(Vadlamudi等人,2004; LeClaire等人)。 ,2008; Narayanan等人,2011; LeClaire等人,2015)。特别地,ARP2亚基的苏氨酸237和238的磷酸化对于允许将ARP2 / 3复合物结构改变为其活性构象是必要的(Narayanan等人,2011; LeClaire等人al ,2015)。虽然对于真核细胞中的许多功能很重要,但ARP2 / 3复合物活性也有利于多种细胞病原体(Haglund和Welch,2011; Welch和Way,2013)。最近,我们证明细菌病原体,嗜肺军团菌,使用注射在宿主细胞质细胞中的细菌蛋白激酶来操纵ARP2 / 3复合磷酸化状态(Michard等人,2015) )。在这里,我们描述如何测试细菌蛋白激酶或另一种蛋白激酶在体外上下文中磷酸化ARP2 / 3复合物的能力。首先,产生和纯化ARP2 / 3复合物和细菌蛋白激酶。然后,将纯化的蛋白质在ATP存在下培养,并通过Western印迹分析ARP2 / ...

2D Diagonal Redox SDS-PAGE of Proteins
Author:
Date:
2013-06-05
[Abstract]  2D diagonal redox SDS-PAGE of proteins is used to detect intramolecular or intermolecular disulfide bridges using Chlamydomonas in this example (Stroeher and Dietz, 2008; Schwarz et al., 2012). Both dimensions consist of a conventional SDS-PAGE, except that the sample buffer for the first dimension lacks a reducing agent. Intermolecular disulfide bridges increase the apparent molecular weight of a protein in the first dimension, whereas intramolecular bridges decrease the apparent weight of the protein. [摘要]  在该实施例中,使用蛋白质的2D对角线氧化还原SDS-PAGE用于检测分子内或分子间二硫键(Stroeher和Dietz,2008; Schwarz等人,2012) 。 两个维度由常规SDS-PAGE组成,除了第一维的样品缓冲液不含还原剂。 分子间二硫桥在第一维中增加蛋白质的表观分子量,而分子内桥降低蛋白质的表观重量。

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