Phos-tag™ Agarose Resins
Phosphorylated proteins can be separated, purified, and concentrated from a mixture using a column filled with Phos-tag™ Agarose. Because it is free of surfactants and reducing agents, phosphorylated proteins can be collected in a condition similar to their in vivo states.
- Phosphorylated proteins can be purified within 1 hour.
- All procedures can be performed under a physiological condition (pH 7.5).
- There is no need for reducing agents or surfactants.
- The procedure is similar to conventional affinity chromatography.
Principles of Affinity Chromatography for Phosphate
Application: Purification of Phosphorylated Protein in A431 Lysate
M: Molecular weight marker
Lane 1: Flow-through fraction
Lane 2: Elution fraction
Lane 3: Washing fraction
A column was filled with Phos-tag™ Agarose, and A431 lysate was applied.
Detection was performed by SYPRO Ruby gel staining (left) and Western blotting using anti-Anti pTyr antibody (right).
Phosphorylated proteins were concentrated in the elution fraction.
- "Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins", E. Kinoshita, A. Yamada, H. Takeda, E. Kinoshita-Kikuta, and T. Koike, J. Sep. Sci., 2005, 28, 155-162.
- "Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH", E. Kinoshita-Kikuta, E. Kinoshita, A. Yamada, M. Endo, and T. Koike, Proteomics, 2006, 6, 5088-5095.
- Exhaustive purification of intercellular phosphorylated proteins using phosphate-affinity chromatography. E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike., Biotechnology Journal, 2007, 7, 217-220.
- "Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates", E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike, Analytical Biochemistry, 2009, 389, 83-85.
- Sample pretreatment technique for high-precision detection of cellular phosphoproteins by using Phos-tag beads. E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, Cell Technology, 2009, 9, 934-938.
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