Ward L D, Simpson R J
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Pept Res. 1991 May-Jun;4(3):187-93.
Polyacrylamide gel electrophoresis is a high-resolving technique for separating proteins. Until recently, its use as a micro-preparative method for isolating microgram amounts of protein in a form suitable for structural analysis was limited by the difficulty in recovering material from the polyacrylamide matrix. This problem has been largely overcome by the development of chemically inert membranes that retain proteins after electroblotting from the polyacrylamide gel. The immobilized proteins are suitable for chemical identification by direct microsequence analysis. Nevertheless, the overall recoveries of proteins using this electrotransfer/blotting approach are low (28%-30%). This paper describes a phase-contrast technique that allows the visualization of proteins during the polyacrylamide gel electrophoresis. This approach is suitable for the quantitative detection of proteins. Since no fixing/staining step is employed, proteins can be recovered in high yield (50%-65%), by passive elution, in a form suitable for microsequence analysis. Methods and strategies for obtaining internal amino acid sequence information from proteins recovered from gels after visualization by high-resolution dynamic imaging are discussed.
聚丙烯酰胺凝胶电泳是一种用于分离蛋白质的高分辨率技术。直到最近，由于从聚丙烯酰胺基质中回收材料存在困难，其作为一种用于分离微克量蛋白质以适合结构分析形式的微量制备方法的应用受到限制。通过开发化学惰性膜，该问题已在很大程度上得到克服，这种膜在从聚丙烯酰胺凝胶电印迹后能保留蛋白质。固定化的蛋白质适用于通过直接微量序列分析进行化学鉴定。然而，使用这种电转移/印迹方法的蛋白质总体回收率较低（28%-30%）。本文描述了一种相差技术，该技术可在聚丙烯酰胺凝胶电泳过程中实现蛋白质可视化。这种方法适用于蛋白质的定量检测。由于未采用固定/染色步骤，通过被动洗脱，可以高产量（50%-65%）回收蛋白质，其形式适合微量序列分析。讨论了从通过高分辨率动态成像可视化后从凝胶中回收的蛋白质获得内部氨基酸序列信息的方法和策略。
