{{'Search' | translate}}
 

PBS, pH 7.4

PBS,pH 7.4

Company: Thermo Fisher Scientific
Catalog#: 10010023
Bio-protocol()
Company-protocol()
Other protocol()

Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
Author:
Date:
2018-10-05
[Abstract]  Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to ... [摘要]  在不同细胞区室之间穿梭蛋白质控制它们的蛋白质稳态,并且在某些情况下可以有助于调节它们的活性。 染色质结合蛋白(例如转录因子)的生化分析通常是困难的,因为它们的产率低并且由于核酸的干扰。 该协议描述了一种有效分离细胞的方法,结合机械(即,超声处理)或酶处理(即,benzonase),有助于分析染色质结合蛋白提取物 通过蛋白质印迹分析或蛋白质下拉分析。 该方法对于富集特定亚细胞级分内的特定蛋白质以研究特定的翻译后修饰模式或鉴定特定的蛋白质 - 蛋白质相互作用可能是有价值的。
【背景】许多染色质结合蛋白的活性和翻译后调节研究很少,因为在分离它们进行生化分析时存在技术困难。这甚至是转录因子的情况,例如基本的螺旋 - 环 - 螺旋(bHLH)转录因子,其通常在组织或细胞模型中具有稀缺的时间和空间表达模式(Dennis 等。,2018)。当生物材料的量成为研究分子途径的障碍时,协议细化有助于解除技术限制(Gillotin和Guillemot,2016)。在我们最近的研究中,我们努力了解神经元bHLH转录因子Ascl1的蛋白水解是如何在神经元分化的细胞模型中调节的(Gillotin et ...

Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection
Author:
Date:
2018-09-05
[Abstract]  Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) stereotaxic injection of the lentivirus into Area X of juvenile zebra finches; 4) analysis of song learning and song performance in juvenile and adult zebra finches. These methods complement the methods employed in recent works that showed changing FoxP2 gene expression in Area X with lentivirus or adeno-associated virus leads to impairments in song behavior. [摘要]  在这里,我们提供了一个详细的逐步协议,用于使用慢病毒操纵幼年斑胸草雀X区的miRNA表达,并分析对歌曲学习和歌曲表现的影响。 该方案有四个部分:1)使慢病毒构建体过表达miRNA miR-9; 2)包装慢病毒载体; 3)将慢病毒立体定位注入少年斑胸草雀X区; 4)青少年和成年斑马雀的歌曲学习和歌曲表演分析。 这些方法补充了近期工作中使用的方法,这些方法显示,在区域X中用慢病毒或腺相关病毒改变 FoxP2 基因表达导致歌曲行为的损害。

【背景】具有良好特征的歌曲行为和基础神经回路的斑胸草雀提供了独特的动物模型来研究声音通信和相关感觉 - 运动学习的神经机制。近年来,一些实验室开始使用病毒载体来操纵斑胸草雀脑中的基因表达并研究其功能后果。这些努力通过对 FoxP2 基因的研究得到了最好的说明,该基因编码叉头盒p2转录因子。 FoxP2蛋白控制着数百个在神经系统发育中起重要作用的下游基因的表达。人类 FoxP2 基因的突变导致言语和语言障碍(Lai et al。,2001)。在鸣禽中,斑马雀X区域的 FoxP2 基因的敲除或过表达,对于声乐学习至关重要的基底神经节核,严重损害了歌曲的行为(Haesler et al。, 2007; Murugan et al。,2013; Heston and ...

Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
Author:
Date:
2018-07-20
[Abstract]  After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow ... [摘要]  在识别细胞外细菌脂多糖(LPS)后,Toll样受体4(TLR4)-CD14信号传导复合物启动两种不同的信号传导途径 - 一种来自质膜,另一种来自信号传导内体(Kagan 等。,2008)。因此,了解TLR4信号转导的早期阶段需要一种稳健且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用,这是LPS检测中最早发生的事件之一。在这里,我们描述了一种基于流式细胞术的方法,我们最近用它来研究离子通道TRPM7在TLR4内吞作用中的作用(Schappe et al。,2018)。该测定依赖于用LPS刺激细胞并使用流式细胞术在不同时间点测量TLR4(或CD14)的细胞表面水平。尽管我们详细描述了来自鼠骨髓来源的巨噬细胞的TLR4和CD14的方法,但它可以很容易地适应于在各种其他信号传导环境中评估受体内吞作用。

【背景】先天免疫细胞,包括巨噬细胞和树突细胞,使用各种模式识别受体(PRR)来调查其环境中的危险和病原体相关分子模式。来自各种亚细胞区室的PRR的贩运和信号传导实现了更广泛的免疫监视,并且已成为先天免疫的重要设计原则(Brubaker et al。,2015)。细菌内毒素LPS的检测高度依赖于TLR4及其共同受体CD14。 TLR4复合物的内吞作用需要CD14,并且对于LPS诱导的巨噬细胞活化是必需的(Zanoni 等人,2011; Tan ...

Comments