Klepsch Mirjam, Schlegel Susan, Wickström David, Friso Giulia, van Wijk Klaas J, Persson Jan-Olov, de Gier Jan-Willem, Wagner Samuel
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Methods. 2008 Oct;46(2):48-53. doi: 10.1016/j.ymeth.2008.06.017. Epub 2008 Jul 30.
In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.
在生物膜中，许多蛋白质以复合物的形式存在。对这些复合物亚基进行整体分析的首选方法是二维蓝色天然（2D BN）/SDS-PAGE。在第一维中，复合物通过BN-PAGE分离，在第二维中，其亚基通过SDS-PAGE分离。在目前可用的方案中，第一维BN凝胶泳道在转移到第二维分离凝胶的过程中会变形。这导致二维BN/SDS凝胶的重现性低且变化大，使其不适用于比较分析。我们开发了一种二维BN/SDS-PAGE方案，其中第一维BN凝胶浇铸在GelBond PAG膜上。固定化可防止BN凝胶泳道变形，从而降低变化并大大提高二维BN/SDS凝胶的重现性。具有固定化第一维的二维BN/SDS-PAGE用于对过表达膜蛋白的大肠杆菌细胞的细胞质膜蛋白质组进行比较分析，并创建了一个包含来自165个不同蛋白斑点的143种已鉴定蛋白质的大肠杆菌细胞质膜蛋白质组的二维BN/SDS-PAGE参考图谱。
