Barnouin Karin
Department of Biochemistry and Molecular Biology, University College London and Ludwig Institute for Cancer Research, London, England, UK.
Methods Mol Biol. 2004;261:479-98. doi: 10.1385/1-59259-762-9:479.
Advances in genomic and proteomic technologies combined with molecular and cell biology have together enabled the identification of numerous genes and their products. Two-dimensional gel electrophoresis (2DE) is especially useful in the study of protein-protein interactions as it permits an improved separation of proteins as well as the detection of specific interacting protein isoform(s) of a protein resulting from post-translational modification. The investigation of interacting proteins using 2DE can be complemented by identification of the proteins by mass spectrometry. Here, I describe how protein complexes, isolated by methods such as immunoprecipitation, can be analyzed by 2DE using either isoelectric focusing (tube gels or immobilized pH gradient strips) or nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension, SDS-PAGE in the second dimension, and gel staining (silver and Coomassie) or Western blotting for the final detection of the interacting proteins.
基因组学和蛋白质组学技术的进步，再结合分子生物学和细胞生物学，共同促成了众多基因及其产物的鉴定。二维凝胶电泳（2DE）在蛋白质 - 蛋白质相互作用研究中特别有用，因为它能更好地分离蛋白质，还能检测蛋白质经翻译后修饰产生的特定相互作用蛋白异构体。使用2DE对相互作用蛋白进行研究时，可以通过质谱鉴定蛋白质来加以补充。在此，我将描述如何通过免疫沉淀等方法分离得到的蛋白质复合物，利用二维凝胶电泳进行分析，即第一维采用等电聚焦（管状凝胶或固定化pH梯度条）或非平衡pH梯度电泳（NEPHGE），第二维采用SDS - PAGE，最后通过凝胶染色（银染和考马斯亮蓝染色）或蛋白质印迹法来最终检测相互作用蛋白。
