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Bromophenol blue

溴酚蓝

Company: Bio-Rad Laboratories
Catalog#: 1610404
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An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
Author:
Date:
2018-02-20
[Abstract]  Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and ... [摘要]  甲醛交联广泛用于与染色质免疫沉淀(ChIP)相结合来测量沿着DNA的相对位置以及转录因子(TF)-DNA相互作用的体内相对水平。但是,通常所做的测量不能提供关于这些交互的动态属性的明确信息。我们已经开发了一种方法来评估来自时间依赖性甲醛交联数据的结合动力学参数,称为交联动力学(CLK)分析。酵母细胞的培养物与甲醛交联不同的时间段,在特定位点产生相对的ChIP信号。我们使用质量作用CLK模型来拟合数据,以提取TF-染色质相互作用的动力学参数,包括开关速率和交联速率。从停车费和停车费中我们可以获得停车和停车时间。以下方案是该方法的第二次迭代,CLKv2,更新了改进的交联和淬火条件,更多关于交联速率的信息以及对观察到的动力学模型建模的系统程序。已应用CLKv2分析来研究TATA结合蛋白(TBP)和其他TF的选定子集的结合行为。该协议使用酵母细胞开发,但也可适用于来自其他生物体的细胞。

【背景】转录起始是一个复杂的过程,涉及染色质化启动子上数十种蛋白的协作和协调相互作用(Kim等人,2005; Encode Consortium,2012; Rhee等人, ,2012; Dowen等人,2014年)。许多研究已经研究了体外核心转录机器的组装和调控(Zawel和Reinberg,1992; Conaway和Conaway,1993; Roeder,1996; ...

Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
Author:
Date:
2017-12-05
[Abstract]  Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein interactions (Duggal and Emerman, 2012). The potency of the IFN signaling response determines the outcome of TBFV infection (Best, 2016; Carletti et al., 2017). Interestingly, data from our lab show that TBFV replication is significantly restricted in cells of the reservoir species Peromyscus leucopus thereby suggesting a potent antiviral response (Izuogu et al., 2017). We assessed the relative contribution of IFN ... [摘要]  蜱传黄热病病毒(TBFV)的细胞感染导致干扰素(IFN)信号传导途径的激活和随后称为IFN刺激基因(ISG)(Schoggins等人,2011)的众多基因的上调。许多ISG通过蛋白质 - 蛋白质相互作用以广泛或特定的方式起作用来防止病毒发病(Duggal和Emerman,2012)。 IFN信号反应的效力决定了TBFV感染的结果(Best,2016; Carletti等人,2017)。有趣的是,我们实验室的数据显示TBFV复制在储库物种Peromyscus leucopus的细胞中显着受到限制,从而表明有效的抗病毒应答(Izuogu等人,2017)。我们评估干扰素信号对抗性的相对贡献。通过敲低IFN反应途径中的主要转录因子来抑制白血病。信号转导和转录激活因子1(STAT1)是专门针对在P。 leucopus细胞通过shRNA技术。我们进一步测试了基因敲低对细胞对IFN反应和限制病毒复制的能力的影响;结果表明当STAT1表达被改变时,leucopus细胞对IFN刺激的反应降低,并且对TBFV复制显着更敏感。

【背景】IFN信号是抵抗侵入宿主细胞的黄病毒的第一道防线(Robertson等人,2009; Lazear和Diamond,2015)。通过模式识别受体(PRR)检测与病毒颗粒相关的分子标记,然后通过转录因子引发下游信号从细胞释放1型IFN(Kawai ...

Cell-free Generation of COPII-coated Procollagen I Carriers
Author:
Date:
2017-11-20
[Abstract]  The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers. [摘要]  该协议的目的是在无细胞反应中产生COPII包被的前胶原I(PC1)载体。 COPII包被的PC1载体由供体膜,胞质溶胶,纯化的重组COPII蛋白和核苷酸重构。 该方案描述了供体膜和细胞溶胶的制备,反应的组装,以及复制的COPII包被的载体的分离和检测。 该无细胞反应可用于测试刺激或抑制PC1包装成COPII包被的载体的条件。
【背景】外壳蛋白复合体II(COPII)在从内质网(ER)途径到高尔基体的运输中起着至关重要的作用。来自ER的货物运输所需的基因在酵母的基因研究中被发现,并且借助于添加有纯化组分的无细胞囊泡萌芽反应来阐明囊泡出芽所需基因的蛋白质产物的精确作用(Novick 1981; Kaiser等人,1990; Barlowe等人,1994)。开发了类似的反应来检测COPII在培养的哺乳动物细胞中来自ER的货物运输中的作用(Kim等人,2005)。哺乳动物COPII包被的囊泡直径大约为80-100nm,似乎太小以至于不能容纳诸如刚性的300nm前胶原I(PC1)三股螺旋杆的大分泌性货物。尽管可能的尺寸差异,COPII对于包括PC1在内的大型货物的分泌是必不可少的(Boyadjiev等人,2006)。最近,我们报道了通过随机光学重建显微镜(STORM),相关光电子显微镜(CLEM)和活细胞成像(Gorur ...

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