Improved comparative proteome analysis based on two-dimensional gel electrophoresis

被引:23
作者
Eravci, Murat
Fuxius, Sandra
Broedel, Oliver
Weist, Stephanie
Eravci, Selda
Mansmann, Ulrich
Schluter, Hartmut
Tiemann, Joachim
Baumgartner, Andreas
机构
[1] Charite, Dept Radiol & Nucl Med Radiochem, D-12200 Berlin, Germany
[2] A&M Proteome Sci, Berlin, Germany
[3] Univ Munich, Dept Med Informat Biometry & Epidemiol, D-80539 Munich, Germany
[4] Charite, Dept Internal Med 4, D-12200 Berlin, Germany
关键词
power analysis; quantification; rat brain; two-dimensional gel electrophoresis;
D O I
10.1002/pmic.200600648
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The purpose of this study was to test the ex-tent 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 mu g and 250 mu g protein, respectively. Only 72 spots were higher (p < 0.05), while 58 spots were significantly lower in the 250-mu g 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.
引用
收藏
页码:513 / 523
页数:11
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