We describe an improved Phos-tag SDS-PAGE (Zn2+-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn2+-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn2+-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn2+-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn2+-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.
