Chapter title |
A Simple Method for Detecting Phosphorylation of Proteins by Using Zn2+-Phos-Tag SDS-PAGE at Neutral pH
|
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Chapter number | 25 |
Book title |
Protein Gel Detection and Imaging
|
Published in |
Methods in molecular biology, August 2018
|
DOI | 10.1007/978-1-4939-8745-0_25 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8744-3, 978-1-4939-8745-0
|
Authors |
Gaurav Kumar, Kumar, Gaurav |
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. |
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