用氯化盐制备质膜囊泡及其作为细胞膜模拟物在膜蛋白相互作用的生物物理特性分析中的应用。
Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions.
机构信息
Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States.
出版信息
Anal Chem. 2012 Oct 16;84(20):8650-5. doi: 10.1021/ac301776j. Epub 2012 Oct 3.
Abstract
Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT. We characterize the size distribution of the produced vesicles. We also show that these vesicles can be used for the biophysical characterization of interactions between membrane proteins.
摘要
质膜衍生小泡被用作研究膜蛋白和膜组织的生化和生物物理特性的模型系统。最广泛使用的泡囊化程序依赖于甲醛和二硫苏糖醇（DTT），但这些活性化学物质可能会在实验结果中引入假象。在这里，我们描述了一种使用含有氯化物盐且不含甲醛或 DTT 的高渗泡囊化缓冲液来泡囊化中国仓鼠卵巢（CHO）细胞的方法，CHO 细胞被广泛用于表达重组蛋白。我们描述了这种方法并对产生的小泡的大小分布进行了特征描述。我们还表明，这些小泡可用于膜蛋白相互作用的生物物理特性分析。
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