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Phosphate Affinity Chromatography     - Enrichment of Phosphorylated Proteins from Cell Lysate -
Phos-tag™ Agarose

Phos-tag™ is a novel phosphate-binding tag at neutral pH (physiological pH) and was developed by Department of Functional Molecular Science at Hiroshima University.   Phos-tag™ technologies are innovative methods using Pho-tag™ Agarose for separation, purification and enrichment of phosphorylated substances.
This method is based on immobilized metal affinity chromatography (IMAC) using a phosphate-binding tag molecule (a dinuclear zinc(II) complex) attached on a highly cross-linked agarose (Phos-tag™ Agarose).

Phos-tag™ Agarose zinc(II)-bound form

Phos-tag™ Agarose is manufactured by MANAC Incorporated.
Phos-tag™ Agarose AG-503

1. Features

  • Purification of Phosphorylated protein in within 1 hour
  • All procedures at physiological pH (pH 7.5)
  • No reducing agents or detergents are necessary.
  • The procedure for Phos-tag™ Agarose is almost the same as that for the general affinity chromatography.

2. Principle of phosphorylation of Affinity Chromatography

Principle of phosphate affinity chromatography

3. Application: Purification of phorphorylated protein in A431 lysate

M: Molecular Weight Marker
Lane 1: Flow-through fraction
Lane 2: Elution fraction
Lane 3: Washing fraction
Place Phos-tag™ Agarose into a column. Apply A431 cell lysate on it. By detections of SYPRO Ruby gel staining (left figure) and Western blotting using Anti pTyr (right figure), phosphorylated proteins were confirmed to enrich in the elution fraction.

4. Troubleshootings

  1. While the volume of the Phos-tagtrade; agarose gel bed is fixed at 1 mL in the protocol, it can be easily optimized in proportion to the amount of the sample.
    Before the pH measurement for the buffer solutions, the pH-electrode system should be calibrated using the two pH buffer solutions (e.g., pH 4 and 7).
  2. When a larger amount of the lysate proteins (e.g., 0.60 mg of A431 cell's proteins in 0.25 mL RIPA buffer) were applied on the 1-mL gel bed, some of the phosphoproteins were eluted in the flow-through and washing fractions. A similar leak into the fractions resulted from the use of twice the volume of an RIPA buffer (i.e., 0.50 mL, 0.50 mg lysate proteins) and the 1-mL gel bed, which may be attributed to the competitive binding of HOVO32- or zinc(II)-elimination by EDTA.
  3. Some proteins adsorb on the centrifugal filter unit.  Please estimate the recovery of the eluted proteins by a protein quantification method (e.g., Bradford method).

5. Product List

Product Name Package Size Wako Catalog No. Grade Storage Condition
    Phos-tag™ Agarose
0.5 mL
Keep at 2-10°C.
3 mL

6. References on Phos-tag™ Chemistry

  • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule, Rapid Communications of Mass Spectrometry, 17, 2075-2081 (2003), H. Takeda, A. Kawasaki, M. Takahashi, A. Yamada, and T. Koike 
  • Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex, Dalton Transactions, 1189-1193 (2004), E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro, and T. Koike
  • Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule, Journal of Lipid Research, 45, 2145-2150 (2004), T. Tanaka, H. Tsutsui, K. Hirano, T. Koike, A. Tokumura, and K. Satouchi
  • Production of 1,2-Didocosahexaenoyl Phosphatidylcholine by Bonito Muscle Lysophosphatidylcholine/Transacylase , Journal of Biochemistry, 136, 477-483 (2004), K. Hirano, H. Matsui, T. Tanaka, F. Matsuura, K. Satouchi, and T. Koike
  • Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins, Journal of Separation Science, 28, 155-162 (2005), E. Kinoshita, A. Yamada, H. Takeda, E. Kinoshita-Kikuta, and T. Koike
  • Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule, Analytical Chemistry, 77, 3979-3985 (2005), K. Inamori, M. Kyo, Y. Nishiya, Y. Inoue, T. Sonoda, E. Kinoshita, T. Koike, and Y. Katayama
  • Phosphate-binding tag: A new tool to visualize phosphorylated proteins, Molecular & Cellular Proteomics, 5, 749-757 (2006), E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, and T. Koike
  • Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH, Proteomics, 6, 5088-5095 (2006), E. Kinoshita-Kikuta, E. Kinoshita, A. Yamada, M. Endo, and T. Koike
  • Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis , Analytical Biochemistry, 360, 160-162 (2007), S. Yamada, H. Nakamura, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and Y. Shiro
  • Label-free kinase profiling using phosphate-affinity polyacrylamide gel electrophresis, Molecular & Cellular Proteomics, 6, 356-366 (2007), E. Kinoshita-Kikuta, Y. Aoki, E. Kinoshita, and T. Koike
  • A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike (The phosphate group at DNA-terminal is efficiently captured by Zn2+.Phos-tag.)
  • Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule Journal of Biomolecular Techniques, 18, 278-286 (2007), T. Nakanishi, E. Ando, M. Furuta, E. Kinoshita, E. Kikuta-Kinoshita, T. Koike, S. Tsunasawa, and O. Nishimura
  • A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE, Proteomics, 8, 2994-3003 (2008), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, S. Yamada, H. Nakamura, Y. Shiro, Y. Aoki, K. Okita, and T. Koike
  • FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway, Nature Structural & Molecular Biology, 15, 1138-1146 (2008), M. Ishiai, H. Kitao, A. Smogorzewska, J. Tomida, A. Kinomura, E. Uchida, A. Saberi, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, S. Tashiro, S. J. Elledge, and M. Takata
  • Two-dimensional phosphate affinity gel electrophoresis for the analysis of phosphoprotein isotypes, Electrophoresis, 30, 550-559 (2009), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, Y. Aoki, S. Ohie, Y. Mouri, and T. Koike
  • Formation of lysophosphatidic acid, a wound-healing lipid, during digestion of cabbage leaves, Bioscience, Biotechnology, and Biochemistry,73, 1293-300 (2009), T. Tanaka, G. Horiuchi, M. Matsuoka, K. Hirano, A. Tokumura, T. Koike, and K. Satouchi
  • A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides, Analytical Biochemistry, 388, 235-241, (2009), K. Takiyama, E. Kinoshita, E. Kinoshita-Kikuta, Y. Fujioka, Y. Kubo, and T. Koike
  • Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates, Analytical Biochemistry, 389, 83-85, (2009), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose, Proteomics, 9, 4098- 4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Ujihara, and T. Koike
  • Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE, Nature Protocols, 4, 1513-1521 (2009), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag, Rapid Communications in Mass Spectrometry, 24, 1075-1084 (2010), J. Morishige, M. Urikura, H. Takagi, K. Hirano, T. Koike, T. Tanaka, and K. Satouchi
  • Genotyping and mapping assay of single-nucleotide polymorphisms in CYP3A5 using DNA-binding zinc(II) complexes, Clinical Biochemistry, 43, 302-306 (2010), E. Kinoshita, E. Kinoshita-Kikuta, H. Nakashima, and T. Koike
  • The DNA-binding activity of mouse DNA methyltransferase 1 is ragulated phosphorylation with casein kinase 1σ/ε, Biochemical Journal, 427, 489-497 (2010), Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and I. Kameshita

7. LINK to Protocol

Protocol - Version 8
Enrichment of Phosphorylated Proteins from Cell Lysate - Phosphate Affinity Chromatography using Phos-tag™ Agarose -
  PDF (1.8 MB/11 p)   (May, 2015)

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