Mauro Sergio, Colignon Bertrand, Dieu Marc, Delaive Edouard, Raes Martine
Département Sciences du Vivant, Centre wallon de Recherches agronomiques, Chaussée de Charleroi 234, 5030, Gembloux, Belgique,
Methods Mol Biol. 2015;1295:427-40. doi: 10.1007/978-1-4939-2550-6_30.
Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post-translational modifications (PTMs) detection.A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.
基于二维荧光差异凝胶电泳（2D-DIGE）的定量二维凝胶蛋白质组学变得可行，并且这项技术已被广泛接受，因为它消除了凝胶间的差异，并极大地促进了在许多不同实验条件下凝胶间的定量比较。然而，几种蛋白质在同一位点的共迁移仍然是一个主要限制，这降低了比较定量的准确性，并妨碍了明确的翻译后修饰（PTM）检测。一种基于传统聚丙烯酰胺凝胶IEF样品分级分离，随后进行连续两次SDS-PAGE电泳的方案减轻了共迁移限制。在提议的方法中，使用两种不同的缓冲系统进行SDS-PAGE是关键。
