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Phos-tag™ Precast Gel
SuperSep Phos-tag™

This is a precast gel copolymerized with Phos-tag™ acrylamide. It can be used immediately after opening the package. Use of this product allows isolation of phosphorylated proteins depending on the phosphorylation level. In addition, it shows excellent storage stability by the use of a neutral gel buffer similarly to SuperSep and gives sharp bands.
It requires neither phospho-specific antibody nor radioactive isotope labeling.


Product Information

⇒ Leaflet (2.1 MB / 4 p)


Phos-tag™ PAGE GUIDEBOOK


⇒ Proteomics Research 2012

1. Principle




2. Features


  • Ready-to-use; Your precious time is saved!
  • Isolation of phosphorylated proteins depending on the phosphorylation level
  • Good isolation with sharp bands
  • Possible to store for a long time (6 months)
  • Requires neither phospho-specific antibody nor radioactive isotope labeling


3. Number of publications citing the use of the Phos-tag™ SDS-PAGE method since 2008




Searched by Google Scholar

4. Examples of Use


(1) Separation of phospho/dephospho-Protein


(2) Time Course of Dephosphorylation





[Electrophoresis buffer]
  Tris-glycine-SDS buffer for electrophoresis

[Electrophoresis samples]
  Lane 1: Non-treated albumin
  Lane 2: Dephosphorylated albumin

[Electrophoresis conditions]   20 mA, 70 minutes
[Staining]   Quick CBB staining
[Decoloration]   Deionized water

Albumin (Wako Cat. No. 010-17071) was dephosphorylated using alkaline phosphatase (NIPPON GENE CO., LTD., Code No. 319-02661).
The result confirmed dephosphorylation by the band shift.


[Electrophoresis buffer]
   Tris-glycine-SDS electrophoresis buffer

[Electrophoresis samples]
  Lane 1: β-casein (AP-treated, 0 minute)
  Lane 2: β-casein (AP-treated, 15 minutes)
  Lane 3: β-casein (AP-treated, 30 minutes)
  Lane 4: β-casein (AP-treated, 45 minutes)
  Lane 5: β-casein (AP-treated, 60 minutes)

[Electrophoresis conditions]    35 mA, 60 minutes)
[Staining]   Quick CBB staining
[Decoloration]   Deionized water

β-casein was dephosphorylated in time series. The results confirmed separation of phosphorylated and dephosphorylated β-caseins. Time-dependent dephosphorylation level cwas also onfirmed.

5. Applications of Phos-tag SDS PAGE


(1) Search for phosphorylation site of Cdk5 (cyclin-dependent kinase 5)-activated sub-unit p35
          using Ala substitution variant

Regarding p35 known phosphorylation sites Ser8 and Thr138, 3 Ala substitution variants were produced (Ser8: S8A, Thr138: T138A, Ser8 and Thr138:2A). These and wild-type p35, as well as Cdk5 or kinase-negative Cdk5, which has no kinase activity, were discovered in the COS-7 cells. The cellular extract was detected by Western blotting using Phos-tag™ SDS-PAGE. (Detected extract: anti-p35 antibody)

Relationship between phosphorylation site and band shift was clarified.

- From lanes 1 (L2, L4) and 5 (M1): p35 is phosphorylated, depending on Cdk5.
- From lanes 1 (L2, L4) and 3 (L2, L4): With about half of p35, Thr138 is phosphorylated at kinase-negative Cdk5,
    and Thr138 is also phosphorylated by kinase other than Cdk5.
- From lanes 5 (M1) and 6 (L3, L4): Ser8 and Thr138 are main phosphorylation sites.
- From lanes 5 (M1), 7 (L1, L2) and 8 (M2): M1 is the phosphorylation site for Ser8 and Thr138. M2 is the phosphorylation
    site for Ser8 only. L1 and L2 are the phosphorylation sites for Thr138 only.
* X in L1, L3: not yet identified
* L4: non-phosphorylated p35

Data published in:
Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag™ SDS-PAGE, T. Hosokawa, T. Saito, A. Asada, K. Fukunaga, and S. Hisanaga, Mol. Cell. Proteomics, Jun 2010; 9: 1133 - 1143.

Data provided by:
Dr. T. Hosokawa, Laboratory for Memory Mechanisms Neural Circuit Function Research Core, Brain Science Institute, RIKEN and Dr. S. Hisanaga, Molecular Neuroscience Laboratory, Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University.

(2) Determining fraction containing kinase for phosphorylating Dnmt1

We were able to determine the fraction that contained the target kinase.

[1] GST-Dnmt1(1-290) bonding protein was obtained from mouse brain extract using affinity chromatography.
[2] Proteins were eluted through the DNA cellulose column by 0.3 M and 1 M NaCl.
[3] In vitro kinase assay was performed in each fraction with GST-Dnmt1(1-290) as substrate.
[4] Kinase activity in the fraction was confirmed by shift band, by Western blotting using Phos-tag™ SDS-PAGE (Detection:Anti mouse Dmnt1 (72-86))

Data published in:
"The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1δ/ε", Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike and I. Kameshita, Biochem. J., May 2010; 427(3): 489-97.

Data provided by: Dr. Y. Sugiyama, Laboratory of Molecular Biology, Science Research Center, Kochi University and Dr. I. Kameshita, Department of Life Science, Faculty of Agriculture, Kagawa University.

(3) Application in two-dimensional eletrophoretic migratio:
          Analysis of phosphorylated form of hnRNPK Dnmt1

hnRNP K was isolated by immunoprecipitation from nuclear homogenate of mouse macrophage cell line J774.1 cells stimulated with LPS, and hnRNP K isoforms were separated using IPG strip gel (pH 4.7-5.9) in the first dimension and Phos-tag™ SDS-PAGE in the second dimension.  Each isoform and modification site was then identified using mass spectrometry.

Each phosphorylated form was distinguished at the same isoelectric point, respectively
(e.g. spots 6 vs. 8 and spots 4 vs. 7).

Data published in: "Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis", Y. Kimura, K. Nagata, N Suzuki, R. Yokoyama, Y. Yamanaka, H. Kitamura, H. Hirano, and O. Ohara, Proteomics, Nov 2010; 10(21): 3884-95.
Data provided by:
Dr. Y. Kimura and Dr. H. Hirano, Yokohama City University and O. Ohara, RCAI, RIKEN.

6. Notes on preparation


  • Sample preparation

    Phos-tag™ SDS-PAGE is vulnerable to contaminant in protein samples, especially to chelating reagent, vanadic acid, inorganic salts, surfactants.
    Cleaning them up by TCA precipitation, dialysis or desalting is strongly recommended before Phos-tag SDS-PAGE.

  • Pre-treatment for transfer

    An additional procedure, elimination of zinc ions (Zn2+) from the gel using EDTA, is necessary before transfer.
    This procedure increases transfer efficiency of proteins from a gel to a membrane.
    1. Prepare 1x transfer buffer with 10 mmol/L EDTA and without EDTA.
    2. Soak the gel in 1x transfer buffer with 10 mmol/L EDTA for a minimum of 20 minutes with gentle agitation. Repeat it 3 times with buffer exchanges.
    3. Soak the in 1x transfer buffer without 10 mmol/L EDTA for 10 minutes with gentle agitation.
    4. Transfer the proteins from the gel to a membrane*.
      * A Wet-tank method is strongly recommended for effective protein transfer.

7. Quality Control


Every batch of SuperSep Phos-tag™ is tested for compliance with its product specification. It is checked that certain phosphorylated and dephosphorylated proteins are separable and the degrees of the proteins' mobility are constant in a practice test.

8. Product List


(1) for Bio-Rad's Electrophoresis Tank  

Product Name Wako Cat. No. Pkg. Size Electrophoresis Tank
SuperSep Phos-tag™ (50 μmol/L), 7.5%, 17 wells
83 x 100 x 3.9 mm
5 gels
Mini-PROTEAN® Tetra Cell
(Bio-Rad Laboratories, Inc.)
SuperSep Phos-tag™ (50 μmol/L), 12.5%, 17 wells
83 x 100 x 3.9 mm
5 gels
Mini-PROTEAN is a registered trademark of Bio-Rad Laboratories, Inc.

(2) for Life Technologies' Electrophoresis Tank 

Product Name Wako Cat. No. Pkg. Size Electrophoresis Tank
SuperSep Phos-tag™ (50 μmol/L), 7.5%, 17 wells
100 x 100 x 6.6 mm
5 gels
XCell SureLock™ Mini-Cell
(Life Technologies, Inc.)
SuperSep Phos-tag™ (50 μmol/L), 12.5%, 17 wells
100 x 100 x 6.6 mm
5 gels
XCell SureLock is a registered trademark of Life Technologies, Inc.

(3) for Wako's Electrophoresis Tank   "EasySeparator"

Product Name Well Wako Cat. No. Pkg. Size Electrophoresis Tank
SuperSep Phos-tag™ (50 μmol/L), 6%
13 wells
5 gels
(Wako Cat. No. 058-07681)
17 wells
5 gels
SuperSep Phos-tag™ (50 μmol/L), 7.5%
13 wells
5 gels
17 wells
5 gels
SuperSep Phos-tag™ (50 μmol/L), 10%
13 wells
5 gels
17 wells
5 gels
SuperSep Phos-tag™ (50 μmol/L), 12.5%
13 wells
5 gels
17 wells
5 gels
SuperSep Phos-tag™ (50 μmol/L), 15%
13 wells
5 gels
17 wells
5 gels
SuperSep Phos-tag™ (50 μmol/L), 17.5%
13 wells
5 gels
17 wells
5 gels

9. Related Products


(1) Wako's Electrophoresis Tank

Product Name Wako Catalog No. Package Size Grade
EasySeparator (An electrophoresis tank for SuperSep precast gels)
1 unit
for Electrophoresis

(2) Precast Gels - SuperSep

Product Name Well Wako Catalog No. Package Size Grade Storage Condition
SuperSep Ace, 6%
13 wells
10 gels
for Electrophoresis
Keep at 2-10°C.
SuperSep Ace, 7.5%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 10%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 12.5%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 15%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 5-12%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 5-20%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 10-20%
13 wells
10 gels
17 wells
10 gels
SuperSep Ace, 15-20% (Tricine Gel)
13 wells
10 gels
17 wells
10 gels

Phos-tag™ Acrylamide™
Phos-tag™ Acrylamide™ shown on a brochure "Proteomics Research 2012"
Phos-tag™ Series

10. FAQ


Q1. Which gel staining methods can we use?
A1. Any staining methods used normally such as CBB staining, negative staining, silver staining and fluorescent staining can be used.

Q2. Decoloration is poor after CBB staining.
A2. Use of a microwave oven for decoloration gives a satisfactory result.
Method: Transfer the stained gel in 100 mL of deionized water, add a few sheets of Kimwipe rounded, and heat in a microwave oven for a few minutes. Change deionized water and repeat the procedure 3 or 4 times. Be careful that the container is hot.

Q3. Can we use the product for the western blotting?
A3. Yes, provided that zinc in the gel is eliminated with EDTA to improve transcription efficiency to membranes.
Method: Immerse the gel in a transcription buffer (25 mmol/L tris, 192 mmol/L glycine, 10% MeOH) containing 10 mmol/L EDTA and agitate gently for 10 minutes. Repeat the procedure 3 times. Then, agitate in a transcription buffer (25 mmol/L tris, 192 mmol/L glycine, 10% MeOH) not containing EDTA for 10 minutes and transcribe onto PVDF membrane or nitrocellulose membrane. If the transcription efficiency is poor, change the condition such as increasing the number of EDTA treatment or increasing EDTA concentration.

Q4.Bands are distorted.
A4. Samples containing EDTA, inorganic salts, surfactants, etc. may ause distortion of bands and tailing. Desalt samples by precipitation with TCA or dialysis.
An empty lane also causes distortion. Apply a sample buffer (x1) in the empty lane.

Q5. Phosphorylation and dephosphorylation of target proteins are not separated.
A5. Perform electrophoresis of β-casein as a positive control and alkaline phosphatase-treated β-casein as a negative control and check the band shift. If there is a band shift, phosphorylation and non-phosphorylation of the target protein may not be separated at the concentration of Phos-tag™ or acrylamide concentration in this product.

Q6. Can we use crude extracts of cells?
A6. Yes, though the Rf value may small and isolation of bands may be obscure depending on the protein targeted.

Q7. How much sample should be applied to each well?
A7.1 to 5 μg in case of purified proteins (CBB staining), 10 to 30 μg in case of tissue or cell extracts (adjust depending on the amount of protein expressed).
* These are recommended amounts. Perform an ordinary SDS-PAGE or western blotting beforehand to determine an appropriate sample size.

Q8.Which molecular markers should we use?
A8.No marker recommended. The molecular weight of a marker is not reflected in this product. Therefore, E. coli -derived recombination protein or dephosphorylated sample is recommended as a negative control instead of a marker.

Q9.What complex ion is it in the product?
A9. Zinc ion.
Q10.How can we know whether the band shift is due to phosphorylation?
A10. Perform electrophoresis with 12.5% SuperSep™ Ace (Wako Catalog No. 199-14971), which has the same gel concentration, and check if the target protein is degraded.




Listed products are for research use only. Do not administer each to human.

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