title: Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling.
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.
authors.affiliation: Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D051176:beta Catenin / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D011994:Recombinant Proteins; D051176:beta Catenin; D016875:tau Proteins; D010434:Pepsin A