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Prefractionation of protein samples for proteome analysis using reversed‐phase high‐performance liquid chromatography
Author(s) -
Badock Volker,
Steinhusen Ulrike,
Bommert Kurt,
Otto Albrecht
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200108)22:14<2856::aid-elps2856>3.0.co;2-u
Subject(s) - chromatography , chemistry , two dimensional gel electrophoresis , proteome , mass spectrometry , acetonitrile , silver stain , resolution (logic) , electrophoresis , high performance liquid chromatography , gel electrophoresis , elution , reversed phase chromatography , coomassie brilliant blue , reproducibility , gel electrophoresis of proteins , proteomics , staining , polyacrylamide gel electrophoresis , biochemistry , microbiology and biotechnology , enzyme , biology , genetics , artificial intelligence , computer science , gene
We describe an approach for fractionating complex protein samples prior to two‐dimensional gel electrophoresis using reversed‐phase high‐performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed‐phase chromatography and elution with a five‐step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high‐resolution two‐dimensional gel electrophoresis (2‐DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2‐DE gels from two different cell states. In addition, this method is suitable for enriching low‐abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.