School of Pharmacy, Wenzhou Medical College, Zhejiang, PR China.
Electrophoresis. 2010 Jan;31(2):411-20. doi: 10.1002/elps.200900472.
A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.
本文描述了一种 SDS-PAGE 上蛋白质的阴性检测方法。在该方法中，曙红 Y（EY）通过与蛋白质形成稳定的水溶性蛋白-染料复合物选择性地沉淀在凝胶背景中，而蛋白质所在的区域则不存在这种复合物。使用 EY 对蛋白质进行负染，可实现高灵敏度、低成本和简单的方案。新描述的方法完成整个方案不到一个小时，检测限为 0.5ng 单条蛋白质带。与咪唑-锌负染相比，EY 染料提供了更宽的线性动态范围、更高的灵敏度和重现性，以及更好的蛋白质带或斑点与背景之间的明显对比度。此外，这里开发的新技术为同时处理多个凝胶提供了一种实际可行的方法，使得高通量染色成为可能，用于蛋白质组学研究。此外，我们还通过 PMF 比较了染色方法对质谱质量的影响。
