Phos-tag^™ Agarose Resins

• 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.

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.

1. "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.
2. "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.
3. Exhaustive purification of intercellular phosphorylated proteins using phosphate-affinity chromatography. E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike., Biotechnology Journal, 2007, 7, 217-220.
4. "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.
5. 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.