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For the purification of phosphorylated proteins on gel chromatography

Phos-tag Agarose Resins

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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.

Features

  • 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

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Application: Purification of Phosphorylated Protein in A431 Lysate

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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.

References

  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.

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