Purification of phosphorylated proteins/peptides by affinity chromatography

Phos-tag Agarose

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This product is agarose bound to Phos-tagTM, a functional molecule that traps phosphate by having two divalent metal ions.
Phosphoproteins can be separated, purified, and concentrated from the mixture by filling the column. Since surfactants and reducing agents are not used, phosphoproteins in a state close to that in vivo can be obtained.
This product is a renewed product with less nonspecific binding than the previous products (302-93561, 308-93563) by adjusting the binding amount of Phos-tagTM functional group.

Features

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  • Phosphoproteins can be purified in less than 1 hour.
  • The proteins can be trapped in physiological condition (pH 7.5).
  • Purified without reducing agent and surfactant.

Data

Application Data

Purification of phosphorylated proteins in A431 lysate

  1. Phos-tag™ Agarose was filled into the column and A431 cell lysate was appliqued onto it.
  2. Elution fractions obtained after adsorption, washing, and elution operations were separated by SDS-PAGE along with cell lysate and flow-through/wash fractions.
  3. Fluorescent staining (detecting all proteins) and Phos-tag™ biotin confirmed that phosphoproteins were enriched in the eluted fraction.
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References

  1. Kinoshita, E. et al.: J. Sep. Sci., 28, 155(2005).
    Novel immobilized zinc (II) affinity chromatography for phosphopeptides and phosphorylated proteins
  2. Kinoshita‐Kikuta, E. et al.: Proteomics, 6(19), 5088(2006).
    Enrichment of phosphorylated proteins from cell lysate using a novel phosphate‐affinity chromatography at physiological pH
  3. Kinoshita, E. et al.: Biotechnol. J., 7, 217(2007). (Japanese)
    リン酸基親和性クロマトグラフィーを用いた細胞内のリン酸化タンパク質の網羅的精製
  4. Kinoshita-Kikuta, E. et al.: Anal. Biochem., 389(1), 83(2009).
    Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates
  5. Kinoshita, E. et al.: Cell Technol., 9, 934(2009). (Japanese)
    細胞内リン酸化タンパク質を高精度に検出するためのPhos-tagビーズによるサンプル前処理法

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Phos-tag™ Biotin

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For research use or further manufacturing use only. Not for use in diagnostic procedures.

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