Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers.
Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers.
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DOI：
10.1021/ac201768k
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发表时间：
2011-10-15
影响因子：
7.4
通讯作者：
Cremer, Paul S.
中科院分区：
文献类型：
--
作者：
Liu, Chunming;Monson, Christopher F.;Yang, Tinglu;Pace, Hudson;Cremer, Paul S.
开发了一种电泳-电渗聚焦 (EEF) 方法，用于根据膜结合蛋白和带电脂质的电荷大小比从最初均质的混合物中分离它们。 EEF 使用相反的电泳力和电渗力将蛋白质和脂质聚焦并分离成支持的脂质双层 (SLB) 上的窄带。膜相关物种以高度可重复的方式集中到 SLB 内的特定位置。通过调节缓冲液 pH、离子强度、电场和温度等实验条件，可以预测和控制蛋白质的稳态聚焦位置。仔细调整变量应该能够分离出只有细微差别的膜蛋白混合物。 EEF 技术被发现是分离低初始浓度蛋白质混合物的有效方法，并且它克服了扩散峰展宽，允许在 380 μm 宽的隔离支撑膜片内同时分离四个条带。
An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch.
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