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Diethyl pyrocarbonate

焦碳酸二乙酯(DEPC)

公司名称: Sigma-Aldrich
产品编号: D5758
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RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis
Author:
Date:
2018-07-05
[Abstract]  RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the ... [摘要]  RNA免疫沉淀(RIP)是一种基于抗体的技术,用于绘制体内 RNA-蛋白质相互作用。 DBR1是一种RNA脱支酶,负责套索RNA的脱支,用于套索RNA的降解和转换。众所周知,主要miRNA(Pri-miRNA)被主要由DCL1和HYL1组成的切割复合物识别并进一步加工成成熟miRNA。由于pri-miRNA的丰度较低,RIP随后的qRT-PCR已被广泛用于评估切割复合物与pri-miRNA在先前研究中的结合效率。因此,缺乏对具有pri-miRNA的切割复合物的全基因组评估。随着高通量测序技术的改进,我们成功地使用RIP-seq比较了Dicing复合物与野生型和 dbr1-2 突变体之间的pri-miRNA的结合效率。 。在该方案中,我们提供了在两种不同基因型之间的HYL1-YFP和DCL1-YFP转基因植物中使用GFP捕获珠的RIP-seq的详细描述。该方法可用于评估pri-miRNA与拟南芥中的切割复合物的结合,并且它可以应用于植物中的其他RNA结合蛋白。

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
Author:
Date:
2017-05-20
[Abstract]  RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic ... [摘要]  RNA代谢在不同的组织和发育阶段被严格控制,其失调是癌症的分子特征之一。 通过直接结合特定的序列元件,RNA结合蛋白(RBP)在共转录和转录后调控事件中起关键作用。 我们最近证实,在胰腺癌细胞中,获得耐药(DR) - 表型取决于多聚嘧啶区结合蛋白(PTBP1)的上调,其又被引入丙酮酸激酶前mRNA并有利于剪接 致癌性PKM2变体。 在这里,我们描述了紫外(UV)光交联和免疫沉淀(CLIP)方法的逐步方案,以确定RBP与贴壁人细胞系中其目标RNA的特定区域的直接结合。

背景 在细胞核中转录时,新生的RNA立即与被称为RNA结合蛋白(RBP)的反式因子立即组装。这些因子直接与RNA分子中特定的顺式调控序列相互作用,从而形成核糖核蛋白(RNP)复合物(Dreyfuss et al。,2002; ...

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
Author:
Date:
2017-03-20
[Abstract]  Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from ... [摘要]  核糖体失活蛋白(RIP)是由于其N-糖基化酶(EC 3.2.2.22)活性而不可逆地灭活核糖体的酶。酶从28S rRNA的腺嘌呤编号4324和大鼠核糖体中的核糖(或来自其他生物体的敏感核糖体中的等同腺嘌呤)切割N-糖苷键。该腺嘌呤位于α-原丝素 - 蓖麻毒素环(SRL)中,这对于在原核生物和真核生物中的mRNA-tRNA易位期间分别在核糖体上锚定延伸因子(EF)G和EF2至关重要。 RIP主要从植物中分离出来,这些蛋白质的实例是蓖麻毒蛋白或者口服抗病毒蛋白(PAP)。这些蛋白质,单独或作为免疫毒素的一部分,是癌症治疗的有用工具。以下方案描述了当通过在聚丙烯酰胺凝胶上电泳在来自兔网织红细胞裂解物的RIP处理的脱嘌呤RNA在酸性苯胺存在下孵育时检测释放的RNA片段的方法。发布的片段(Endo的片段)是RIP的动作的诊断。

Endo和Tsurugi首先在大鼠核糖体中描述了RIP对真核28S rRNA的N-糖基化酶活性,蓖麻毒素(Endo and ...

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