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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Humans
  • Hydrogen-Ion Concentration
  • Pepsin A / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Recombinant Proteins / metabolism
  • Swine
  • beta Catenin / metabolism
  • tau Proteins / metabolism


  • Phosphoproteins
  • Recombinant Proteins
  • beta Catenin
  • tau Proteins
  • Pepsin A