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

AbstractWe 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 Bis‐tris‐buffered neutral‐pH gel system to detect shifts in the mobility of phosphoproteins. An existing 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 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 Zn2+–Phos‐tag SDS‐PAGE gels showed better long‐term stability than the Mn2+–Phos‐tag SDS‐PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate‐affinity SDS‐PAGE.