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Thermal stabilization of tissues and the preservation of protein phosphorylation states for two‐dimensional gel electrophoresis
Author(s) -
Smejkal Gary B.,
RivasMorello Chiara,
Chang JaeHyung Robert,
Freeman Emily,
Trachtenberg Alexander J.,
Lazarev Alexander,
Ivanov Alexander R.,
Kuo Winston P.
Publication year - 2011
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.201100170
Subject(s) - phosphorylation , phosphoprotein , dephosphorylation , phosphatase , kinase , chemistry , protein phosphorylation , biochemistry , enzyme , microbiology and biotechnology , in vivo , gel electrophoresis , protein kinase a , biology
2‐DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal p I shifts were a common feature in 2‐D gels. Of the 588 matched proteins separated on 2‐D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p <0.05). Phosphoprotein‐specific staining was corroborated by the identification of 16 phosphoproteins by nano‐LC MS/MS and phosphotyrosine kinase activity assay.