A Simple Method for Detecting Phosphorylation of Proteins by Using Zn²⁺-Phos-Tag SDS-PAGE at Neutral pH

Abstract

Zn²⁺-Phos-tag SDS-PAGE at neutral pH is a novel and simple method for analysis and separation of phosphorylated forms of proteins from their nonphosphorylated forms. This technique exploits the use of a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olate which acts as a phosphate-binding tag, having the capacity to incorporate two zinc metal ions which could then bind to phosphomonoester dianion as a bridging ligand. The acrylamide-pendant Zn²⁺-Phos-tag provides a phosphate affinity on simple SDS-PAGE gel for detection of mobility shift in phosphorylated proteins as compared to their nonphosphorylated forms. The technique is based on the principle that Zn²⁺-Phos-tag bound phosphorylated protein has a slower migration rate on the gel as compared to unbound nonphosphorylated proteins and are thus separated on the gel. Zn²⁺-Phos-tag SDS-PAGE was developed by improving the Mn²⁺-Phos-tag SDS-PAGE as the latter was unsuccessful in showing a mobility shift in some proteins such as Tau and pepsin. Additionally, the use of neutral pH instead of alkaline pH gives almost about 6 months of stability to the gels as compared to gels in alkaline pH which were quite unstable. Therefore, this Zn²⁺-Phos-tag SDS-PAGE method is simple, reliable and convenient for phosphate-affinity SDS-PAGE.