Abstract
Phosphorylation is a major regulatory mechanism controlling protein and cell function. Phosphoproteomics is continuing to reveal the extent and complexity of protein phosphorylation. In particular, most proteins are emerging to contain multiple phosphorylation sites. However, phosphoproteomics has outpaced current understanding of the functional roles of individual phospho-sites. In this paper the Phos-tag gel method is presented and discussed in the context of other available tools for phosphorylation research. Strengths and weaknesses of Phos-tag gels are outlined and an integrated approach to phosphorylation research is proposed. SignificanceThe Phos-tag gel method has unique strengths especially regarding the analysis of multi-site phosphorylation. A combined approach including Phos-tag gels together with other methods like isotope labelling, phospho-specific antibodies, and mass spectrometry is required to advance current understanding of protein phosphorylation.
Highlights
Phos-tag gels are used to analyze the phosphorylation status of proteins
Phos-tag acrylamide, a derivative of the phosphate capture molecule conjugated to acrylamide, can be included into polyacrylamide gels to reduce the electrophoretic mobility of phosphorylated proteins compared to their non-phosphorylated counterparts
The polyacrylamide-immobilized Phos-tag complex leads to slower migration of phosphoproteins during electrophoresis by reversibly binding to phosphate moieties on them
Summary
This is the peer reviewed version of the following article: Nagy, Z., Comer, S., & Smolenski, þÿA. (2018). This is the peer reviewed version of the following article: Nagy, Z., Comer, S., & Smolenski, þÿA. Analysis of protein phosphorylation using Phos tag gels. The UCD community has made this article openly available. Please share how this access benefits you. Analysis of protein phosphorylation using Phos-tag gels Zoltan Nagy, Shane Comer and Albert Smolenski. The Phos-tag technology provides a simple small-scale approach to study protein phosphorylation and is compatible with traditional vertical slab minigel systems. By offering information on the ratio of phosphorylated versus non-phosphorylated protein species, Phos-tag gels enable comprehensive characterization of protein phosphorylation. The technology is becoming increasingly popular because it can complement studies using phosphorylation site-specific antibodies or phosphoproteomics, where information on phosphorylation site occupancy is not readily available
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