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High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics
Author(s) -
Cong WeiTao,
Hwang SunYoung,
Jin LiTai,
He HongZhang,
Choi JungKap
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900472
Subject(s) - proteome , staining , chromatography , detection limit , chemistry , stain , silver stain , linear range , proteomics , mass spectrometry , protein detection , throughput , microbiology and biotechnology , biochemistry , biology , nanotechnology , materials science , computer science , gene , telecommunications , genetics , wireless
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.