Phos-tag SDS-PAGE for the analysis of milk phosphoproteins involved in cow’s milk allergy

Abstract

Phos-tag functions as a phosphate-binding tag molecule in an aqueous solution under near-physiological conditions. Its affinity for a divalent phosphate ion is 16,000 times greater than that for a monovalent carboxylate ion at neutral pH. We have developed and applied useful techniques for the analysis of phosphoproteins based on Phos-tag. Among these, this chapter presents a phosphate affinity technique for the analysis of phosphoproteins by electrophoresis using Phos-tag. Our electrophoretic method using SDS-PAGE, which is widely used for molecular weight-based separation of proteins, allowed us to separate and detect phosphoproteins and non-phospho counterparts on an identical SDS-PAGE gel. Here we describe the resolving power of Phos-tag SDS-PAGE for the separation and detection of milk phosphoproteins, α-casein and β-casein, as typical protein samples. This technique would have a major impact not only on the analysis of milk phosphoproteins involved in cow’s milk allergy but also on the analysis of all food phosphoproteins.