Williams Taufika Islam, Combs Jennifer C, Thakur Anup P, Strobel Herbert J, Lynn Bert C
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0286, USA.
Electrophoresis. 2006 Jul;27(14):2984-95. doi: 10.1002/elps.200500730.
A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
一种基于二乙醇胺-N,N-双(2-羟乙基)甘氨酸（Bicine）的新型十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）缓冲系统被开发用于膜蛋白分析。该方法涉及在二维中对完全变性和溶解的蛋白质进行基于分子量的分离。这种双SDS-PAGE（dSDS-PAGE）方法产生了蛋白质斑点的对角线排列，并成功规避了与涉及传统基于凝胶方法的膜蛋白质组分析相关的问题。来自厌氧细菌嗜热栖热放线菌（Clostridium thermocellum）的膜蛋白用于这些研究。将三羟甲基氨基甲烷-双SDS-PAGE（Tricine-dSDS-PAGE）和新开发的二乙醇胺-N,N-双(2-羟乙基)甘氨酸-双SDS-PAGE（Bicine-dSDS-PAGE）与标准甘氨酸-双SDS-PAGE（Laemmli方案）在分离嗜热栖热放线菌膜蛋白的适用性方面进行了比较。使用优化的凝胶制备和运行缓冲液条件进行的大幅面凝胶实验表明，与甘氨酸-双SDS-PAGE相比，Tricine-dSDS-PAGE的蛋白质斑点数量增加了112%，Bicine-dSDS-PAGE增加了151%。数据清楚地表明，二乙醇胺-N,N-双(2-羟乙基)甘氨酸-双SDS-PAGE是分析膜蛋白的一种优越方法，提供了更高的分辨率和蛋白质呈现。
