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Phos-tag MG-Bead Technical Information
Phos-tag MG-Bead is Magnetic beads to separate phosphorylated biomolecules.
Phos-tag MG-Bead provides an efficient procedure for separation of phosphorylated biomolecules including native phosphopeptides from biological samples at physiological pH. The phosphate enrichment procedure needs an appropriate magnet unit and buffers for the binding, washing, and elution processes.
Citation
How to Use
Phosphorylated biomolecules are bound to Phos-tag MG-Bead and separated by using magnetic stand.
Application Data
Enrichment of Phosphorylated peptides
| Bead volume | 50 µL (Phos-tag MG-bead: FMN-binding site ≈ 3 nmol/µL-gel) |
|---|---|
| Sample | Tryptic digest of 5 nmol β-casein in Sol. A (0.10 mL) 1P is the monophosphopeptide and 4P is the tetraphosphopeptide. |
| Washing buffer | Sol. B (0.20 mL x 3) and dist. water (0.20 mL x 1) |
| Elution buffer | Sol. E (0.10 mL x 3) |
| How to measure | The total time for the phosphate-affinity column chromatography was within 12 min. All the fractions are analyzed by HPLC using a reverse-phase column. |
# Both phosphopeptides are efficiently separated from nonphosphorylated peptides.
# The presence of neutral salt NaCl ensures complete elimination of nonphosphorylated peptides.
# The recoveries of 1P and 4P in the elution fraction E1 are 69% and 71%, respectively.
# The total recoveries of P1 and P4 in the E1 – E3 fractions are more than 95%.
# The obtained phosphorylated peptides can be desalted and condensed using a hydrophobic resin such as C18-ZipTip (Milipore).
Separation of NAD and its 2’-phosophorylated counterpart NADP
| Bead volume | 50 µL (Phos-tag MG-bead: FMN-binding site ≈ 3 nmol/µL-gel) |
|---|---|
| Sample | Mixed solution of 100 nmol NAD and 100 nmol NADP in Sol. A (0.10 mL) |
| Washing buffer | Sol. A (0.20 mL x 3) and dist. water (0.20 mL x 1) |
| Elution buffer | Sol. E (0.10 mL x 5) |
| How to measure | The total time for the phosphate-affinity column chromatography was within 12 min. All the fractions are analyzed by HPLC using a reverse-phase column. |
# The experimental result shows a distinct separation of NADP from NAD.
# The total recovery of the eluted NADP in E1– E5 fractions are more than 99%.
