Premium
Assessing factors for reliable quantitative proteomics based on two‐dimensional gel electrophoresis
Author(s) -
Fiévet Julie,
Dillmann Christine,
Lagniel Gilles,
Davanture Marlène,
Negroni Luc,
Labarre Jean,
de Vienne Dominique
Publication year - 2004
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.200300731
Subject(s) - coomassie brilliant blue , proteomics , staining , yeast , electrophoresis , chromatography , two dimensional gel electrophoresis , gel electrophoresis , biochemistry , biology , chemistry , genetics , gene
Abstract We statistically analysed various factors to get accurate estimates of protein quantities from two‐dimensional gels. Yeast proteins were labelled with 35 S or stained with Coomassie Brilliant Blue G‐250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With 35 S‐labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue‐stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains.