Minden Jonathan S
Mellon Institute, Carnegie Mellon University, Pittsburgh, PA, USA.
Methods Mol Biol. 2012;869:287-304. doi: 10.1007/978-1-61779-821-4_24.
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, difference gel electrophoresis (DIGE) is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over a ∼20,000-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 days 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，这些染料设计为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。使用合适的成像系统，差异凝胶电泳（DIGE）能够在约20000倍的蛋白质浓度范围内可靠地检测低至0.2飞摩尔的蛋白质，以及低至±15%的蛋白质差异。因此，DIGE与数字图像分析相结合大大改善了蛋白质组变异的统计评估。在此，我们描述了一个进行DIGE实验的方案，该方案需要2至3天完成。
