High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation
High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation
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DOI：
10.1016/j.ab.2011.01.038
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发表时间：
2011-05-15
影响因子：
2.9
通讯作者：
Yamashita, Atsuko
中科院分区：
文献类型：
--
作者：
Ihara, Makoto;Matsuura, Noriko;Yamashita, Atsuko
建立了一种改进的非变性聚丙烯酰胺凝胶电泳（PAGE）方法，该方法能够评价膜蛋白的流体动力学状态并允许凝胶内荧光检测。在该方法中，双（烷基）磺基琥珀酸酯用于为洗涤剂溶解的膜蛋白提供负电荷，以促进适当的电泳迁移，而不干扰其天然流体动力学状态。该方法实现了高分辨率的电泳分离，在良好的协议，通过尺寸排阻色谱得到的洗脱曲线。标记的绿色荧光蛋白（GFP）的凝胶内荧光检测的适用性，便于分析样品，而无需任何纯化。这种方法可能作为一个通用的分析技术，用于评估膜蛋白的折叠，寡聚化，和蛋白质复合物的形成。(C)2011 Elsevier Inc. All rights reserved.
An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins. (C) 2011 Elsevier Inc. All rights reserved.