Danos Olivier, Svinartchouk Fedor
UMR 8115 Généthon, Evry, France.
Electrophoresis. 2006 Sep;27(17):3475-9. doi: 10.1002/elps.200500036.
2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.
双向电泳（2-DE）是蛋白质组学研究中的一项重要工具。然而，2-DE固有的凝胶间差异常常掩盖了样本之间的生物学差异，并影响蛋白质表达水平的定量比较。在此，我们描述了一种对2-DE的改进方法，该方法可提高蛋白质的匹配度和定量准确性。这是通过在由透析膜隔开的两条固相pH梯度（IPG）胶条中对两个样本进行等电聚焦（IEF）来实现的。IEF运行后，将胶条分开，并在两块单独的凝胶上进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）。用考马斯亮蓝（CBB）对凝胶进行染色后，使用ImageMaster 2D Platinum软件（安玛西亚公司）进行斑点检测和定量分析。用该方法对C2C12成肌细胞的蛋白质提取物进行分析，结果显示斑点匹配效率达到99%，98%的斑点染色强度（%体积）的变异系数（CV）小于0.5。我们得出结论，与在单独胶条上进行的IEF相比，这种称为透析辅助凝胶电泳的技术具有更优的斑点匹配度和定量重现性。
