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

  • Gaurav Kumar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)

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.

Key words

Phos-tag Zinc(ii) metal ion SDS-PAGE Phosphorylated protein Nonphosphorylated protein Mobility shift 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Oklahoma Medical Research Foundation, University of OklahomaOklahoma CityUSA

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