Highly resolving two-dimensional gels for protein sequencing.

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort. IMAGES:

以载体两性电解质进行第一维分离的二维聚丙烯酰胺凝胶电泳（two-dimensional polyacrylamide gel electrophoresis, 2D PAGE），已成为同步分析细胞蛋白质的成熟工具。为将2D PAGE的应用范畴拓展至制备级层面，我们探究了固定化pH梯度（immobilized pH gradients, IPG）应用于第一维分离的可行性。所得实验结果表明，单块IPG凝胶可承载多达1 mg的细胞蛋白质，且不会造成分辨率损失。此前在基于载体两性电解质的二维凝胶中检出的突变多肽，在基于IPG的二维凝胶中同样可被有效检出。借助IPG凝胶，仅需10块凝胶即可分离得到数百种细胞多肽，其产量足以满足当前主流测序技术的测序需求。因此我们认为，IPG技术极大地提升了细胞蛋白质大规模测序的应用前景，可为构建与二维凝胶相关的蛋白质数据库、鉴定新型多肽基因产物提供强力支撑，这对于基因组测序工作亦具有相应的重要意义。图像：