Viswanathan Surya, Unlü Mustafa, Minden Jonathan S
Department of Biological Science, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
Nat Protoc. 2006;1(3):1351-8. doi: 10.1038/nprot.2006.234.
Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as 'spots' with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to +/- 15%, over a >10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 d to complete.
二维差异凝胶电泳（2D DIGE）是二维电泳（2DE）的一种改良形式，它能让人们在同一块凝胶上同时比较两个或三个蛋白质样品。每个样品中的蛋白质都用不同颜色的荧光染料进行共价标记，这些染料设计成在电泳过程中对蛋白质的相对迁移没有影响。样品中共有的蛋白质会以具有固定荧光信号比例的“斑点”形式出现，而样品之间不同的蛋白质则具有不同的荧光比例。借助合适的成像系统，DIGE能够可靠地检测低至0.5飞摩尔的蛋白质，并且在超过10000倍的蛋白质浓度范围内，蛋白质差异低至±15%。因此，DIGE与数字图像分析相结合极大地改善了蛋白质组变异的统计评估。在这里，我们描述了一个进行DIGE实验的方案，该方案需要2 - 3天完成。
