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2 ml microtube

2.0 mL Safe-Lock 离心管

Company: Eppendorf
Catalog#: 0030120094
Other protocol()

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
[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)。 ...

Stable-isotope Labeled Metabolic Analysis in Drosophila melanogaster: From Experimental Setup to Data Analysis
[Abstract]  Stable-isotope labeled metabolic analysis is an essential methodology to characterize metabolic regulation during biological processes. However, the method using stable-isotope-labeled tracer (e.g., 13C-glucose) in live animal is only beginning to be developed. Here, we contribute a qualitative metabolic labeling experiment protocol in Drosophila melanogaster using stable-isotope-labeled 13C-glucose tracer followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Detailed experimental setup, data acquisition and analysis are provided to facilitate the application of in vivo metabolic labeling analysis that might be applied in a wide range of biological studies. [摘要]  稳定同位素标记的代谢分析是表征生物过程中代谢调节的基本方法。 然而,在活体动物中使用稳定同位素标记的示踪剂(例如, 13 C-葡萄糖)的方法才刚刚开始开发。 在这里,我们使用稳定同位素标记的 13 C-葡萄糖示踪剂,然后通过液相色谱 - 质谱(LC-MS)在 Drosophila melanogaster 中提供定性代谢标记实验方案。 分析。 提供详细的实验设置,数据采集和分析以促进体内代谢标记分析的应用,其可以应用于广泛的生物学研究中。

【背景】代谢组学是一项新兴的omic-level研究,旨在研究复杂生物系统中的小分子代谢物。它已被应用于与人类健康和疾病有关的各种研究领域,例如生物标志物发现,疾病发病机理和药物毒性评估。代谢物的测量对于确定响应内源和外源变化的代谢途径的改变是重要的。为了准确表征代谢途径活动,已使用同位素标记的示踪剂(例如, 13 C和 15 N)(Park et al。,2016; Jang et al。,2018)。在培养的细胞中有许多这样的研究(定量和定性)(Buescher et al。,2015; Liu et al。,2018),然而,基于稳定同位素的研究活体动物的代谢标记实验在很大程度上尚未开发。在目前的方案中,我们描述了使用标记的 13 ...

Microscopic Observation of Subcellular GFP-tagged Protein Localization in Rice Anthers
[Abstract]  This protocol demonstrates a simple method to determine the subcellular localization of fluorescence-tagged proteins on the vibratome sections of rice developing anthers. If a cell type-specific promoter is used to drive the tagged protein-encoding gene, the method enables to clearly distinguish the cells retaining fluorescent signals from other anther cells. It is applicable to both live and fixed samples, and presumably to other plant tissues. [摘要]  该方案演示了一种确定荧光标记蛋白在水稻发育花药的振动切片上的亚细胞定位的简单方法。 如果使用细胞类型特异性启动子来驱动标记的蛋白质编码基因,该方法能够清楚地区分保留荧光信号的细胞与其他花药细胞。 它适用于活的和固定的样品,并且可能适用于其他植物组织。