| 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 ...
[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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| Biotinylation of Cell Surface Proteins
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Author:
Date:
2012-05-05
[Abstract] Membrane proteins are major sensors of extracellular stimuli and initiators of intracellular signal transduction, and their abundance on the cell surface in particular is often dynamically regulated even when there are no significant changes of their total abundance in a cell. This protocol is designed to biochemically label and separate membrane proteins on the plasma membrane from those in the intracellular compartments. In conjunction with co-immunoprecipitation and western blot analysis, functional analysis of dynamic interaction of membrane proteins with other membrane proteins or ...
[摘要] 膜蛋白是细胞外的刺激和细胞内信号转导发起的主要传感器，他们大量并特定的存在细胞表面上。它们虽然受到动态调节，但是在细胞中的总量没有显著变化。该方法的目的是给浆膜的膜蛋白打上生化标签，并且在细胞内将它们分离出来。与免疫共沉淀和免疫印迹分析相结合，膜蛋白和其他膜蛋白或者细胞内接头和效应蛋白的动态交互功能分析，都可以实现。
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