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Improved comparative proteome analysis based on two‐dimensional gel electrophoresis
Author(s) -
Eravci Murat,
Fuxius Sandra,
Broedel Oliver,
Weist Stephanie,
Eravci Selda,
Mansmann Ulrich,
Schluter Hartmut,
Tiemann Joachim,
Baumgartner Andreas
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600648
Subject(s) - spots , proteome , difference gel electrophoresis , chromatography , staining , two dimensional gel electrophoresis , gel electrophoresis , silver stain , chemistry , electrophoresis , stain , significant difference , fluorescence , microbiology and biotechnology , proteomics , biology , biochemistry , optics , genetics , statistics , mathematics , physics , gene
The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two‐dimensional electrophoresis gels (pH 4–7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 µg and 250 µg protein, respectively. Only 72 spots were higher ( p <0.05), while 58 spots were significantly lower in the 250‐µg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between‐gel variation. This resulted in the detection of significant differences in spot intensities in 77–90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.