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Tetramethylethylenediamine (TEMED) for electrophoresis

TEMED

Company: Carl Roth
Catalog#: 2367
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Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
Author:
Date:
2021-03-05
[Abstract]  

Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The

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[摘要]  [摘要]非洲锥虫和其他原生动物生物线粒体内的基因表达依赖于核苷酸特异性的RNA编辑反应。在该过程中,仅将尿苷(U)-核苷酸位点特异性插入序列不足的初级转录物中,并从中缺失,以将其转化为可翻译的mRNA。该反应由0.8 MDa的多蛋白复合物催化,该复合物被称为编辑体。在这里我们描述了一种改进的体外试验,以定量探索Editosome的催化活性。该测定使用合成的,荧光团衍生的寡核糖核苷酸 作为编辑底物,可通过耦合到激光诱导荧光(LIF)检测系统的毛细管电泳(CE)自动分离反应产物。该测定法功能强大,只需要纳克级的材料,并且通过使用多毛细管CE / LIF仪器,可以高度平行的方式进行测定。进一步的改进包括使用硫代磷酸酯修饰的,因此具有RNase耐性的底物RNA,以及用于同时监测U插入和U缺失反应的多重型荧光团标记策略。该测定方法对于研究酶体的机理和酶学是有用的。ħ H但是,它也可以在高通量执行以筛选RNA编辑特异性抑制剂。


图形摘要:


基于荧光的体外U插入/ U缺失RNA编辑(FIDE)分析的特征




[背景]中的RNA编辑反应动质体原生动物如非洲锥虫和利什曼原虫表示一个信使的最显着的转录后修饰(米)的RNA(综述Göringer ...

Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis
Author:
Date:
2021-02-05
[Abstract]  

Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing

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[摘要]  [摘要]在过去的十年中,已经注意到,微生物病原体和害虫将小RNA(sRNA)效应子传递到宿主植物中,以操纵植物生理学和免疫力,称为跨界RNA干扰。在此过程中,真菌和卵菌寄生虫sRNA劫持了植物ARGONAUTE(AGO)/ RNA诱导的沉默复合体,以转录后沉默宿主靶基因。我们在此描述方法学的细节,我们如何在宿主植物拟南芥感染期间恢复卵菌病原体拟南芥的跨界sRNA效应子。该生物协议包含两个部分:第一,关于植物AGO / sRNA co- 免疫纯化和sRNA回收,用于Illumina高通量测序分析。其次,我们解释了如何进行生物信息学小号斯尔纳序列分析读取可使用Galaxy服务器。原则上,该协议适用于研究来自多种宿主植物和植物相互作用(微生物)的AGO结合的sRNA。


[背景]小RNA(sRNA)可以充当病原体效应物,劫持植物ARGONAUTE(AGO)/ RNA诱导的沉默复合物(RISC),并使宿主mRNA沉默以进行感染,这种病毒被称为跨界RNA干扰的毒力机制(Weiberg等。,2015; Zeng等,2019)。分析感染期间与植物AGO结合的sRNA的库是一种选择方法,以全面了解可能通过宿主AGO / RISC起作用的植物入侵性病原体sRNA。基于抗体的植物AGO / ...

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 / ...

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