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Mini Trans-Blot® Electrophoretic Transfer Cell

Mini Trans-Blot ®电泳转移池

Company: Bio-Rad Laboratories
Catalog#: 1703930
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Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle
Author:
Date:
2020-04-05
[Abstract]  Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes. These include ultracentrifugation, ultrafiltration, size exclusion chromatography, immune capture and precipitation using a proprietary polymer. Here, we provide a protocol based on differential ultracentrifugation and sucrose density gradients tailored for the isolation of crude and ultra-pure exosomes from primary fibroblast cultures derived from adult mouse skeletal ... [摘要]  [摘要 ] 外来体是几乎所有细胞分泌的动态纳米囊泡,并存在于所有生物体液中。鉴于它们的异质含量很高,外泌体已牵涉到它们通过影响细胞-细胞和细胞-ECM通讯而发挥的许多生理和病理过程。近年来,已经建立了越来越多的方法外泌体的纯化和表征。其中包括超速离心,超滤,尺寸排阻色谱,免疫捕获和使用专有聚合物的沉淀。在这里,我们提供了基于差分超速离心和蔗糖密度梯度的协议,该协议专门用于从成年小鼠骨骼肌衍生的原代成纤维细胞培养物中分离粗制和超纯外泌体。可以对该协议进行修改和修改,以从各种组织和体液中分离和表征外泌体。


背景 ] ë xosomes是单膜,异质纳米囊泡直径范围从30至150nm,secre 由所有细胞和存在于几乎所有的体液泰德。外泌体中存在的可溶性和膜大分子,mRNA,microRNA的光谱取决于代谢状态以及分泌这些纳米囊泡的细胞的发育起源。由于它们的货物组成,外泌体可以启动接收细胞中的信号传导途径,并参与了发育,免疫和正常组织生理的维持。在神经退行性疾病,纤维化和癌症等疾病条件下,它们被证明可以触发和传播病理刺激(Rackov 等,2018; Gurunathan 等,2019; van de Vlekkert 等,2019)。在这里,我们描述了从成年小鼠腓肠肌(GA)肌肉建立的成纤维细胞培养物中纯化外泌体的方案(van de Vlekkert ...

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Author:
Date:
2018-07-05
[Abstract]  Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure ... [摘要]  来自产后啮齿动物的小脑颗粒神经元(CGN)已被广泛用作研究神经元发育,生理学和病理学的模型。 CGN体外培养维持成熟小脑颗粒细胞在体内显示的相同特征,包括发育致密的神经炎网络,神经元活动,神经递质释放和神经元的表达 蛋白质标记。 此外,CGN代表了梭菌神经毒素(CNT)研究的便利模型,最着名的是破伤风和肉毒杆菌神经毒素,因为它们大量表达CNT受体和神经元内基质, ie ,可溶性N-乙基马来酰亚胺 - 敏感因子激活蛋白受体(SNARE蛋白)。 在这里,我们描述了从出生后大鼠/小鼠获得高纯度CGN培养物的方案和用CNT中毒的简便方法。 我们还说明了评估CNT活性及其抑制的方便方法。

【背景】梭菌神经毒素(CNT)的大家族由破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNT)的多种变体形成,它们分别是破伤风和肉毒中毒的神经麻痹毒素(Schiavo et al。,2000; Johnson和Montecucco,2008; Rossetto et al。,2014)。 TeNT,七种BoNT血清型(BoNT / A至/ G)及其许多亚型是金属蛋白酶,通过切割SNARE蛋白(可溶性N-乙基马来酰亚胺敏感因子激活蛋白受体),三种必需蛋白质来阻断神经递质的释放而引起神经麻痹。控制突触小泡与突触前质膜的融合(Rossetto et al。,2014; ...

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; ...

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