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Gilson pipettes

Company: Scientific Laboratory Supplies
Catalog#: F144801
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Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
Author:
Date:
2018-10-05
[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)。 ...

In vitro Enzymatic Assays of Histone Decrotonylation on Recombinant Histones
Author:
Date:
2018-07-20
[Abstract]  Class I histone deacetylases (HDACs) are efficient histone decrotonylases, broadening the enzymatic spectrum of these important (epi-)genome regulators and drug targets. Here, we describe an in vitro approach to assaying class I HDACs with different acyl-histone substrates, including crotonylated histones and expand this to examine the effect of inhibitors and estimate kinetic constants. [摘要]  I类组蛋白去乙酰化酶(HDACs)是有效的组蛋白去蛋白酶,拓宽了这些重要(epi-)基因组调节因子和药物靶标的酶谱。 在这里,我们描述了一种体外方法来测定具有不同酰基 - 组蛋白底物的I类HDAC,包括巴豆酰化组蛋白,并将其扩展以检查抑制剂的作用并估计动力学常数。

【背景】组蛋白的翻译后修饰是基因组调控的重要方面,包括基因表达(例如参见Pengelly et al。,2013;在Castillo 等人中综述,2017)。组蛋白修饰改变染色质结构和/或调节蛋白质的结合,例如核小体重塑因子(在Bannister和Kouzarides,2011中综述)。大多数组蛋白修饰是可逆的并且可以酶促去除。例如,通过组蛋白脱乙酰基酶(HDAC)除去组蛋白乙酰化,其中存在几类。近年来,新的组蛋白赖氨酸酰化,包括琥珀酰化,丙酰化,丁酰化,羟基丁基化和巴豆酰化已成为规范组蛋白乙酰化的新替代物,并且已经证实了许多这些新发现的组蛋白修饰的功能相关性(Sabari 等人,2017)。特别是,组蛋白巴豆酰化与活性基因表达有关,并被认为受细胞代谢状态的影响(Sabari et al。,2015; Fellows et al。 ,2018年)。最近已显示I类组蛋白脱乙酰酶也有效地去除组蛋白质(Wei et al。,2017; Fellows et al。,2018)。
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A Lentiviral Pseudotype ELLA for the Measurement of Antibodies Against Influenza Neuraminidase
Author:
Date:
2018-07-20
[Abstract]  This protocol describes the rapid and safe production of lentiviral pseudotypes characterized by a lentiviral core containing a reporter, in conjunction with avian influenza haemagglutinin (HA) and human neuraminidase (NA) glycoproteins on the surface. Production is optimized with Endofectin LentiTM transfection reagent in 6-well plate format. These pseudotyped viruses can be employed for serological assays of surface glycoproteins HA and NA. They can be efficiently used to perform the ELLA (Enzyme-linked lectin assay) to measure NA inhibiting antibodies in lieu of using reassortant virus or Triton X-100 inactivated wild-type virus as source of antigen, which may require higher biosafety levels. [摘要]  该方案描述了慢病毒假型的快速和安全生产,其特征在于含有报道分子的慢病毒核心,以及表面上的禽流感血凝素(HA)和人神经氨酸酶(NA)糖蛋白。 使用6孔板形式的Endofectin Lenti TM 转染试剂优化生产。 这些假型病毒可用于表面糖蛋白HA和NA的血清学测定。 它们可以有效地用于进行ELLA(酶联凝集素测定)以测量NA抑制抗体,而不是使用重配病毒或Triton X-100灭活的野生型病毒作为抗原来源,这可能需要更高的生物安全水平。

【背景】流感病毒假型的产生先前已被广泛描述(Nefkens et al。,2007; Temperton et al。,2007; Carnell et al。,2015)。需要一种安全快速的系统来评估通过ELLA测定靶向NA的抗体,避免使用重配错配病毒或野生型病毒,这已经通过产生携带NA型流感假型来满足(Prevato et al。,2015)。最近的一项研究(Biuso et al。,2017)证实HA与NA的共表达改善了新形成的假型慢病毒的释放。在这里,我们报告了一种简单,广泛适用和优化的PV生产方案,使用6孔板格式的Endofectin Lenti TM ...

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