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Corning® 15 mL PP Centrifuge Tubes, Bulk Packed with CentriStarTM Cap, Sterile

Corning ® 15mL PP离心管,装有CentriStar TM

Company: Corning
Catalog#: 430791
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A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins
Author:
Date:
2018-09-05
[Abstract]  Many proteins appear exclusively nuclear at steady-state but in fact shuttle continuously back and forth between the nucleus and the cytoplasm. For example, nuclear RNA-binding proteins (RBPs) often accompany mRNAs to the cytoplasm, where they can regulate subcellular localization, translation and/or decay of their cargos before shuttling back to the nucleus. Nucleocytoplasmic shuttling must be tightly regulated, as mislocalization of several RBPs with prion-like domains such as FUS and TDP-43 causes the cytoplasmic accumulation of solid pathological aggregates that have been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Traditionally, interspecies heterokaryon assays have been used to determine whether a nuclear ... [摘要]  许多蛋白质在稳态下仅出现核,但事实上在细胞核和细胞质之间连续地来回穿梭。例如,核RNA结合蛋白(RBP)通常伴随mRNA到达细胞质,在那里它们可以在穿梭回到细胞核之前调节其货物的亚细胞定位,翻译和/或腐烂。必须严格调节核质穿梭,因为几种RBP与朊病毒样结构域如FUS和TDP-43的错误定位导致固体病理性聚集体的细胞质积累,这些聚集体与肌萎缩侧索硬化症(ALS)和额颞叶痴呆等神经退行性疾病有关。 (FTD)。传统上,种间异核体分析已被用于确定感兴趣的核蛋白是否穿梭;这些分析是基于来自两个不同物种(例如,小鼠和人类)的供体和受体细胞之间的融合,可以根据不同的染色质染色模式区分,并检测蛋白质的外观。受体核。然而,异核体的鉴定需要经验并且容易出错,这使得难以获得用于定量研究的高质量数据。此外,荧光标记的RBP在供体细胞中的瞬时过表达通常导致其异常的亚细胞定位。在这里,我们提出定量测定,其中表达接近生理水平的eGFP标记的RBP的稳定供体细胞系与表达膜标记物CAAX-mCherry的受体细胞融合,允许容易地鉴定和成像大量高可信度异核体。我们的测定法可用于测量任何感兴趣的核蛋白在不同细胞类型,不同细胞条件下或突变蛋白之间的穿梭活性。

【背景】要了解蛋白质的各种功能,重要的是找出它在细胞内定位的位置。标准的微观和生物化学方法仅在其稳态浓度高于检测阈值时才揭示蛋白质的存在。他们不排除它在短暂地定位的情况下扮演其他重要角色的可能性(Gama-Carvalho和Carmo-Fonseca,2001)。例如,许多RBP在不同的细胞区室中发挥作用,它们伴随着它们的结合mRNA(通常未检测到)并连接真核基因表达的多个步骤(Müller-McNicoll和Neugebauer,2013)。 ...

Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes
Author:
Date:
2018-08-05
[Abstract]  Since its inception, super-resolution microscopy has played an increasingly important role in the discovery and characterization of nanoscale biological structure. dSTORM, which is one of the most commonly applied methods, relies on stochastic photoswitching of fluorophores to recreate a super-resolution image. The cardiac field has particularly benefitted from the application of this technique, as it has enabled sub-diffraction-limit visualization of calcium release units (CRUs) and the fundamental structures that trigger contraction. Acquisition of such images requires careful, reproducible sample preparation, and consistent imaging conditions maintained for the duration of the experiment. Here we present standardized methods for the production of dSTORM images of the Ca2+ ... [摘要]  自成立以来,超分辨率显微镜在纳米级生物结构的发现和表征中发挥着越来越重要的作用。 dSTORM是最常用的方法之一,它依赖于荧光团的随机光切换来重建超分辨率图像。心脏场特别受益于该技术的应用,因为它已经实现了钙释放单元(CRU)的子衍射极限可视化和触发收缩的基本结构。获取这些图像需要仔细,可重复的样品制备,并且在实验期间保持一致的成像条件。在这里,我们提出了生产心肌细胞中Ca 2 + 释放通道Ryanodine Receptor type-2(RyR2)的dSTORM图像的标准化方法。所提出的方案特别关注涉及原发性心肌细胞分离,样品制备和成像的步骤,其中提供了针对实验溶液和显微镜设置的细节。本讨论之后是各种分析技术的概述,以识别集群和CRU中的RyR2组织

【背景】近年来,超分辨率显微镜的普及率迅速提高。已经描述了各种超分辨率技术,其使光学分辨率远低于光的衍射极限,在某些情况下接近可通过电子显微镜获得的光学分辨率。总之,这些技术的出现导致了纳米级生物结构,结构域和蛋白质相互作用的新研究的爆炸式增长。一种流行的超分辨率技术是直接随机光学显微镜(dSTORM),与标准共聚焦显微镜相比,它将相对简单的样品处理的优势与分辨率提高了约10倍(van de Linde ...

Brain Tissue Culture of Per2::Luciferase Transgenic Mice for ex vivo Bioluminescence
Author:
Date:
2018-07-05
[Abstract]  In circadian research, it is essential to be able to track a biological rhythm for several days with the minimum perturbation for the organisms or tissues. The use of transgenic mice lines, in which the luciferase reporter is coupled to a molecular clock protein (here PERIOD2), gives us the opportunity to follow the circadian activity in different tissues or even single clock cells for days without manipulation. This method creates sections using a mouse brain matrix, which allows us to obtain several brain samples quickly at a single time point. [摘要]  在昼夜节律研究中,能够以最小的生物或组织扰动跟踪生物节律数天是至关重要的。 使用转基因小鼠系,其中荧光素酶报告基因与分子钟蛋白(此处为PERIOD2)偶联,使我们有机会在不经操作的情况下跟踪不同组织或甚至单个时钟细胞中的昼夜节律活动数天。 该方法使用鼠标脑矩阵创建切片,这允许我们在单个时间点快速获得几个脑样本。

【背景】昼夜节律是大约24小时循环的行为或分子变化,并且在没有任何外部线索的情况下持续。在哺乳动物中,运动活动,体温和激素释放是昼夜节律的实例,其在位于下丘脑的视交叉上核(SCN)时钟的控制下。 SCN细胞保持内源性节律的能力是由于时钟基因表达的正负循环组成的分子机制:首先,CLOCK和BMAL1蛋白异二聚化通过E-box位点激活不同基因的转录关于基因如 period ( Per1-3 )和隐花色素( Cry1-2 ; Takahashi)的启动子 et al。,2008)。然后,PERIOD和CRYPTOCHROME的蛋白质异二聚化并返回到细胞核以防止BMAL1与E-Box结合。因此,PERIOD和CRYPTOCHROME抑制其自身的转录(Takahashi et al。,2008)。第二个环由类视黄醇相关的孤儿受体(ROR)和Rev-Erb组成:ROR蛋白激活 Bmal1 基因,而REV-ERB蛋白抑制它通过 ROR反应 Bmal1 ...

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