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Phos‐tag SDS‐PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions
Author(s) -
Kinoshita Eiji,
KinoshitaKikuta Emiko,
Koike Tohru
Publication year - 2012
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.201100524
Subject(s) - tris , chemistry , phosphorylation , molecular mass , polyacrylamide gel electrophoresis , kinase , biochemistry , tcep , agarose , gel electrophoresis , microbiology and biotechnology , chromatography , biology , enzyme , phosphine , catalysis
Abstract We have previously reported a neutral‐pH gel system buffered with Bis‐Tris hydrochloride (Bis‐Tris–HCl) in Zn 2+ –Phos‐tag SDS‐PAGE for advanced profiling of phosphoproteins with molecular masses of 10–200 kDa. In the current work, we describe characteristics of two neutral‐pH gel systems, Bis‐Tris–HCl and Tris–acetic acid (Tris–AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10–200 kDa cellular proteins, the Bis‐Tris–HCl system showed a higher resolving power in a 2‐D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1‐D migration patterns of phosphorylated species of extracellular signal‐regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr‐202 and Tyr‐204, in the two buffer systems at the same concentration of Zn 2+ –Phos‐tag. In contrast, shifts in the mobility of various phosphorylated species of a high‐molecular‐mass protein, ataxia telangiectasia‐mutated kinase (ATM, 350 kDa), could only be detected in the Tris–AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.