| An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
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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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| Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons
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Author:
Date:
2016-05-20
[Abstract] This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the ...
[摘要] 该方案旨在从培养的神经元生物素标记和亲和纯化质膜蛋白。 蛋白质生物素化包括生物素与蛋白质共价连接。 生物素是具有小尺寸（MW 244.31g / mol）的膜不可渗透分子，因此不干扰蛋白质的正常功能。 生物素以高亲和力结合链霉抗生物素蛋白和抗生物素蛋白分子。 这种结合对温度，pH和蛋白水解具有极高的抗性，可以捕获和纯化质膜蛋白。 此外，蛋白质可以结合几个生物素分子，这将允许几个链霉抗生物素蛋白或抗生物素蛋白分子的结合，增加感兴趣的蛋白质的检测灵敏度。 在该方案中，活的培养神经元的细胞表面的蛋白质被生物素化。 制备神经元提取物并用NeutrAvidin偶联的珠收集生物素化的蛋白质，并通过Western印迹进行分析。
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| Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3)
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Author:
Date:
2012-07-20
[Abstract] It has become clear that the post-embryonic growth and development of plants requires properly controlled short distance cell-to-cell communication not only through the historically well-known phytohormones, but also through secreted small peptide signals. This protocol demonstrates an example of how to isolate small peptides (< 10 daltons) from complex protein mixtures (e.g. cauliflower meristem protein extraction) for MS/MS analysis.
[摘要] 已经变得清楚的是，植物的后胚胎生长和发育需要适当控制的短距离细胞与细胞的通信，不仅通过历史上熟知的植物激素，而且通过分泌的小肽信号。 该方案展示了如何从复杂蛋白质混合物（例如花椰菜分生组织蛋白质提取）中分离小肽（<10道尔顿）用于ms/ms分析的实例。
s分析的实例。="">
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