title: A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.
abstract: 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 μL 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.
authors.affiliation: Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Tsunehiro
authors.forename: Yuma
authors.initials: Y
authors.lastname: Meki
authors.forename: Kanako
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008280:Magnetics / Q000295:instrumentation*; D009711:Nucleotides / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D055767:Immobilized Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D014357:Trypsin; D015032:Zinc