A Detailed and Radioisotope-free Protocol for Electrophoretic Mobility Shift Assay (EMSA)
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Author:
Date:
2020-08-20
[Abstract] To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. In the present protocol, DNA probes are labeled with Biotin. Therefore, it is safer for researchers when they do not need to use radioisotope-labeled probes. In addition, this protocol is to provide a detailed procedure for a successful EMSA assay. The interested recombinant protein can be mixed with ...
[摘要] [摘要] 为了全面表征转录因子(TF)的功能,需要分析该TF与目标基因座的相互作用。已经开发了几种方法,例如β-葡萄糖醛酸苷酶(GUS)或荧光素酶报道基因,酵母单杂交(Y1H),染色质免疫沉淀(ChIP )和电泳迁移率变动分析(EMSA)分析。其中,EMSA是体外的 可以证明TF与目标DNA片段之间直接相互作用的方法。在本协议中,DNA探针用生物素标记。因此,当研究人员不需要使用放射性同位素标记的探针时,它更安全。此外,该协议还将为成功的EMSA分析提供详细的程序。可以将感兴趣的重组蛋白与靶向DNA探针混合,以在室温(25°C)下进行结合反应。之后,反应混合物可以在天然聚丙烯酰胺凝胶中运行,并转移到带正电的尼龙膜上。最后,可以通过生物素-链霉亲和素化学发光检测结果并可视化。
[背景 ] EMSA是检测蛋白(之间的直接相互作用的有效方法例如,转录因子)和DNA片段(陈,2011)。通常,与蛋白质结合的DNA探针的移动速度比凝胶中的游离DNA探针的移动速度慢。可以观察到这种蛋白质-DNA复合物的移动带移位,而游离DNA探针向凝胶底部的移动速度更快。基于此特征,可以用放射性同位素[ 例如Phosphorus-32(32 ...
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Immunoprecipitation of Tri-methylated Capped RNA
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Author:
Date:
2018-02-05
[Abstract] Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm ...
[摘要] 蜂窝静止(因此已知为G <子> 0 子>骤停)是由降低的DNA复制,增加自噬表征,并且增加的细胞周期蛋白依赖性激酶p27蛋白上标kip1 表达。静止对伤口愈合,器官再生和瘤形成是必不可少的。先前的发现表明微小RNA(miRNA)在调节细胞静止过程中起重要作用。我们最近的出版物静止期间以实例阐述在原代人替代miRNA生物途径包皮成纤维(HFF)细胞的存在。实际上,我们已经发现了一组与由trimethylguanosine合酶1(TGS1)蛋白的2,2,7- trimethylguanosine(TMG)带肩改性PRI-的miRNA(其成熟miRNA发现静止期期间诱导的)的并运输到细胞质通过Exportin-1(XPO1)蛋白质。我们用来抗体针对(TMG)兴趣盖(不与第(m交叉反应 7 G)兴趣帽了大部分PRI-的miRNA或mRNA的含有[鲁曼等人的,1982]),以从增殖或静态HFFS的总RNA提取RNA进行免疫沉淀。该测定的新颖性是PRI-miRNA的以及含有一个TMG-帽修改以外的非编码RNA的特异性分离。
【背景】蜂窝静止,类型可逆生长停滞的,是在伤口愈合,器官再生,和预防瘤形成涉及一种重要的细胞状态(科勒,2011;瓦尔古等人 2012)。已发现小的非编码RNA如miRNA参与细胞静止的调节。 ...
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Determining Ribosome Translational Status by Ribo-ELISA
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Author:
Date:
2018-01-05
[Abstract] The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules that are isolated by physical fractionation in sucrose gradients or other methods.
[摘要] Ribo-ELISA最初是为阐明基于核糖核苷酸化方法(RPM)的核糖体结合新生链检测的基础而开发的。 Ribo-ELISA能够表征核糖体的翻译状态,并且可以应用于通过蔗糖梯度中的物理分离或其他方法分离的新型核糖体相关大分子的超核糖体复合物的发现。
【背景】核糖体是由40S和60S亚单位组成的异构结构,当多个核糖体与单个mRNA结合时,它们以单体和多核糖体存在于细胞中。 另外,翻译核糖体可以与调节翻译的多个分子复合物相关联。 核糖体ELISA(Enzyme-Linked ImmunoSorbent Assay)能够通过核糖体相关新生链的体外嘌呤化检测翻译核糖体(David等人,2012)。 在将嘌呤霉素添加至具有结合的新生链的核糖体时,这种化学反应自发进行。 该方法定量测定每个核糖体中出现的新生链的数量,并且可以用于确定单核细胞相对于多核糖体的翻译状态,并鉴定结合其他大分子的核糖体,其改变其在沉淀柱中的沉降速率或迁移。
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