Immobilization of the first dimension in 2D blue native/SDS–PAGE allows the relative quantification of membrane proteomes

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• 作者：Mirjam Klepsch ; Susan Schlegel ; David Wickströ ; m ; Giulia Friso ; Klaas J. van Wijk ; Jan-Olov Persson ; Jan-Willem de Gier ; Samuel Wagner
• 关键词：Membrane protein ; Protein complex ; Two-dimensional blue native SDS– ; PAGE ; Comparative proteomics ; E. coli
• 刊名：Methods
• 出版年：2008
• 出版时间：October 2008
• 年：2008
• 卷：46
• 期：2
• 页码：48-53
• 全文大小：382 K

文摘

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.