A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules

作者：Tsunehiro Masaya; Meki Yuma; Matsuoka Kanako; Kinoshita Kikuta Emiko; Kinoshita Eiji^*; Koike Tohru

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2013, 925: 86-94.

DOI：10.1016/j.jchromb.2013.02.039

• Magnetic bead
• Nucleotide
• Phosphate-affinity separation
• Phosphopeptide
• Phos-tag

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 mu L of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol.

• 出版日期2013-4-15