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Quantitative detection of phosphoproteins by combination of two‐dimensional difference gel electrophoresis and phosphospecific fluorescent staining
Author(s) -
Stasyk Taras,
Morandell Sandra,
Bakry Rania,
Feuerstein Isabel,
Huck Christian W.,
Stecher Guenther,
Bonn Guenther K.,
Huber Lukas A.
Publication year - 2005
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.200500026
Subject(s) - fluorescent staining , staining , fluorescence , chromatography , electrophoresis , gel electrophoresis , microbiology and biotechnology , chemistry , biology , genetics , physics , quantum mechanics
Here we combine a standard two‐dimensional difference gel electrophoresis (DIGE) protocol with subsequent post‐staining of gels with phosphospecific fluorescent Pro‐Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2‐DE‐gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.