| Charging State Analysis of Transfer RNA from an α-proteobacterium
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Author:
Date:
2020-12-05
[Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces ...
[摘要] [摘要]转移RNA（tRNA）是遗传密码与蛋白质之间的重要纽带。在翻译过程中，tRNA带有其同源氨基酸，并将其传递至核糖体，因此可作为蛋白质合成的底物。为了分析特定tRNA的电荷状态，纯化总RNA并在酸性尿素凝胶上进行分析。然后将分离的RNA转移到膜上并用目标tRNA的探针进行检测。在这里，我们提出了一种改进的协议来分析α-变形杆菌Rhodopseudomonas palustris中的tRNA充电状态 。与传统方法相比，优化了RNA分离步骤以适合这种生物。另外，非放射性平台用于电泳和RNA印迹。这显着减少了此协议所需的时间和精力。
[背景] tRNA的主要功能是，与其他翻译因素的帮助，以确保mRNA的蛋白质的准确的翻译。氨基酰基-tRNA（带电）将氨基酸带到核糖体中以延长肽段，然后释放不带电荷的tRNA。tRNA的充电状态主要取决于可用资源（即氨基酸）及其被核糖体的消耗量。为了分析细胞tRNA的充电状态，已经开发了使用酸性脲凝胶分离总RNA并通过Northern印迹检测感兴趣的tRNA的方法（Janssen等人，2012； ...
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| Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
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Author:
Date:
2020-12-05
[Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris. Following this protocol, RNA can be extracted from either purified ribosomes or directly from cell cultures, and ribosomal RNAs quantified using Northern blot. This protocol gives an example of studying ribosomes in a bacterium other than the commonly used E. coli. The challenge of performing Northern blots with rRNA is also addressed in detail.
[摘要] [摘要]核糖体是细胞生命的组成部分。它们是由多种核糖体蛋白和RNA组成的复杂分子机器。为了研究核糖体组成的不同方面，几十年来已经开发了许多方法。在这里，我们描述如何从α-变形杆菌Rhodopseudomonas palustris中纯化核糖体。按照该协议，可以从纯化的核糖体中提取RNA，也可以直接从细胞培养物中提取RNA，并使用Northern印迹对核糖体RNA进行定量。该协议给出了研究除常用大肠杆菌外的细菌中核糖体的一个例子。还详细介绍了使用rRNA进行Northern杂交的挑战。
[背景]细菌细胞的命运是紧密相连的核糖体。我们最近的研究表明，活性核糖体在营养缺乏的palustris细胞的存活机制中起着重要作用（Yin等人，2019）。核糖体通过一系列超速离心纯化，并采用经典方法优化的方法（Lawrence等，2016）。使用不太常用的毛细管转移系统，通过RNA印迹检测核糖体RNA群体。纯化步骤的细节可能极大地影响核糖体的状态。这些方法在这里进行了详细描述，对于研究多种细菌中的翻译设备的研究人员应该具有广泛的兴趣。
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| RNA Isolation and Northern Blot Analysis
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Author:
Date:
2014-03-20
[Abstract] The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by ...
[摘要] Northern印迹是在分子生物学研究中用于通过检测样品中的RNA来研究基因表达的技术。 使用Northern印迹，可以在分化，形态发生以及异常或疾病状况期间观察到特定的基因表达水平。 在这里，我们检查ATF3，ATF4和GADD153基因表达谱通过北部污点在Vero细胞和H1299细胞后IBV感染。 在IBV（感染性支气管炎病毒）感染的细胞中提取RNA，并使用电泳分离RNA样品。 通过毛细管转移将RNA从电泳凝胶转移到印迹膜上。 使用与靶序列的一部分互补的杂交探针检测特异性mRNA。 通过RT-PCR制备探针，并使用DIG标记试剂盒通过地高辛（DIG）标记。
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