Phos-tag™ PAGE GUIDEBOOK

Principle and Application

Phos-tag™ is …

Phos-tag™ is a functional molecule that binds specifically phosphorylated ions. It is applicable for the specific separation of phosphorylated proteins (Phos-tag™ Acrylamide) as well as for the detection using western blot (Phos-tag™ Biotin), purification (Phos-tag™ Agarose), and MALDI-TOF/MS (Phos-tag™ Mass Analytical Kit).

【Basic Structure of Phos-tag™
Basic Structure of Phos-tag™
M2+:Zinc ion or manganese ion
◇ Selectivity of binding of a phosphate ion (2-) is much higher than that of other anions.
◇ Stable complex is formed under physiological conditions (pH 5 to 8).
Product Purpose of Use
Phos-tag™ Acrylamide Separation:Separation is possible by SDS-PAGE depending on the degree of phosphorylation.
SuperSep Phos-tag™ Separation:Ready-to-Use Precase gel containing 50 µM Phos-tag™ Acrylamide
Phos-tag™ Biotin Detection:A substitute for the anti-phospho antibody used in western blot.
Phos-tag™ Agarose Purification:Phosphorylated proteins are purified by column chromatography.
Phos-tag™
Mass Analytical Kit
Analysis:This is used in MALDI-TOF/MS analysis to improve the detection sensitivity of phosphorylated molecules.

Phos-tag™ was developed by Department of Functional Molecular Science at Hiroshima University. http://www.phos-tag.com/

Principle

  • Two metallic ions cooperate to bind a phosphate group

    Two metallic ions cooperate to bind a phosphate group
  • Phosphorylated proteins move while being bound by Phos-tag™ in the gel

    Phosphorylated proteins move while being bound by Phos-tag™ in the gel
  • Migration speed of phosphorylated proteins decreases and they are separated from non-phosphorylated proteins.

    Migration speed of phosphorylated proteins decreases and they are separated from non-phosphorylated proteins.

Application ― Time course of phosphorylation by using the Tyrosin Kinase Abl ―

Phosphorylated tyrosine was prepared by GST binding protein of tyrosine kinase Abl and the substrate peptide (Abltide) and separated with conventional SDS-PAGE and Phos-tag™ SDS-PAGE, respectively.

  • Conventional SDS-PAGE(CBB staining)

    Conventional SDS-PAGE(CBB staining)
  • Phos-tag™ SDS-PAGE(CBB staining)

    Phos-tag™ SDS-PAGE(CBB staining)