Enrichment of low-molecular-weight phosphorylated biomolecules using phos-tag tip

Abstract

In this chapter, we provide a standard protocol for phosphate-affinity column chromatography for the separation of phosphorylated and nonphosphorylated biomolecules by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties (Phos-tag Tip) was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphorylated biomolecules, such as phosphopeptides, in the field of neuroscience.