Phos-tag™ PAGE GUIDEBOOK

Principle and Application

Phos-tag™ is …

Phos-tag™ is a functional molecule which captures phosphorylated Ser/Thr/Tyr and His/Asp/Lys. It is a synthesized chemical compound designed from an alkaline phosphatase catalytic domain as a model. A series of reagents utilizing Phos-tag™ can be used in the separation, detection, mass spectrometry analysis, and purification of phosphorylated proteins.

【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 precast 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™ Tip Purification:Ready-to-use tip to purify phosphorylated peptides
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 the 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 protein

    1. Divalent metal ions trap phosphorylated proteins during migration.
    2. The higher the amount of phosphorylation, the slower the migration velocity.
    3. Separation occurs based on phosphorylation levels.
    (Separation can occur if the phosphorylation sites are different, even with identical levels of phosphorylation)

Application - Time-course of α-casein dephosphorylation -

Phos-tag™ SDS-PAGE and conventional SDS-PAGE was used to separate α-casein samples, which have been dephosphorylated over time (incubation duration: 0–120 min) by alkaline phosphatase.

Application  -  Time-course of α-casein dephosphorylation  -