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Proteomics. 2011 Jan;11(2):319-23. doi: 10.1002/pmic.201000472. Epub 2010 Dec 14.

Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling.

Author information

1
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp

Abstract

We describe an improved Phos-tag SDS-PAGE (Zn(2+)-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-tris-buffered neutral-pH gel system to detect shifts in the mobility of phosphoproteins. An existing technique (Mn(2+)-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn(2+)-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant Tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole-cell lysates. Additionally, the Zn(2+)-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn(2+)-Phos-tag SDS-PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate-affinity SDS-PAGE.

PMID:
21204258
DOI:
10.1002/pmic.201000472
[Indexed for MEDLINE]

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