High resolution clear native electrophoresis for In-gel functional assays and fluorescence studies of membrane protein complexes
High resolution clear native electrophoresis for In-gel functional assays and fluorescence studies of membrane protein complexes
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DOI：
10.1074/mcp.m700076-mcp200
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发表时间：
2007-07-01
影响因子：
7
通讯作者：
Schaegger, Hermann
中科院分区：
文献类型：
--
作者：
Wittig, Ilka;Karas, Michael;Schaegger, Hermann
透明天然电泳法和蓝色天然电泳法是分离膜蛋白复合体的微尺度技术。考马斯蓝G-250染料用于蓝色天然电泳法，会干扰凝胶内荧光检测和凝胶内催化活性测定。这个问题可以通过在透明的天然电泳法中省略染料来解决。然而，透明天然电泳法的缺点是在电泳法过程中蛋白质聚集增强，蛋白质条带变宽，因此很少被使用。为了保留这两种电泳法的优点，我们用阴离子和中性洗涤剂的无色混合物取代了蓝色天然电泳法的阴极缓冲液中的考马斯染料。与考马斯染料一样，这些混合胶束对膜蛋白施加电荷移动，以增强其阳极迁移，并在电泳过程中改善膜蛋白的溶解性。这种改进的透明天然电泳法提供了与蓝色天然电泳法相当的膜蛋白复合体的高分辨率。我们证明了高分辨率透明天然电泳法用于测定线粒体复合体I-V的凝胶内催化活性的优越性。我们提出了第一个呼吸复合体III的凝胶内组织化学染色方案。此外，我们还展示了高分辨率透明天然电泳法在检测由活性荧光染料标记的荧光标记蛋白和由荧光蛋白标记的荧光标记蛋白方面的独特优势。高分辨率清晰自然电泳法的优势使该技术在功能蛋白质组学分析中具有优势。
Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.