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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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