Phos-tag™ PAGE GUIDEBOOK

Application Data and References

Application Data 1

Impressions of users of Phos-tag™ Acrylamide are introduced herein. We received data and comments from Dr. Tadayuki Ogawa of the University of Tokyo. In addition, we received data applied to two-dimensional electrophoretic migration from Dr. Yayoi Kimura of Yokohama City University, as well as Western blotting applied data from Dr. Yasunori Sugiyama of Kochi University and Dr.Tomohisa Hosokawa of RIKEN.

“I recommend Phos-tag™.” Tadayuki Ogawa, Graduate School of Medicine, the University of Tokyo Phos-tag™ is a very convenient reagent that can be applied in a variety of samples and research purposes. It allows quantitative analysis not only of in vitro assay samples but also in vivo samples in a phosphorylated state. Phos-tag™ SDS-PAGE utilizes normal electrophoretic migration and does not require the purchase of special equipment, so you could say it has good cost performance. Phosphorylation research that used to require anti-phosphorylated antibodies, RI, and many other reagents will now be advanced with Phos-tag™.

Comparison of proteins phosphorylated using Phos-tag™ SDS-PAGE and non-phosphorylated proteins
Comparison of proteins phosphorylated using Phos-tag™ SDS-PAGE and non-phosphorylated proteins

The kinase-reacted phosphorylated proteins in (2) - (5) show clear differences compared with the non- phosphorylated protein in (1).
Data such as the quantitative ratio of phosphorylated and non-phosphorylated proteins, degree of phosphorylation, and population distribution can be readily obtained from band shifts and density.
(Source: Graduate School of Medicine, the University of Tokyo)

Application Data 2
Application in two-dimensional electrophoretic migration: Analysis of phosphorylated forms of hnRNPK

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.

2D electrophoretic migration

Each phosphorylated form was distinguished at the same isoelectric point, respectively.
(eg: 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.

Application Data 3
Determining fraction containing kinase for phosphorylating Dnmt1

Western

  • 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))

“We were able to determine the fraction that contained the target kinase.”
  • Data published in:
    The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1delta/epsilon. 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.

Application Data 4
Search for phosphorylation site of Cdk5-activated sub-unit p35 using Ala substitution variant

Cdk5: cyclin-dependent kinase 5

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)

Western

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

Relationship between phosphorylation site and band shift was clarified!
  • 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.

References

Regarding Phos-tag™ reagents:
  • 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phos phate capture molecule, Rapid Communications of Mass Spectrometry, 17, 2075-2081 (2003),H. Takeda, A. Kawasaki, M. Takahashi, A. Yamada, and T. Koike

  • 2. Phosphate-binding tag: A new tool to visualize phosphorylated proteins, Molecular & Cellular Proteomics, 5, 749-757 (2006),E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, and T. Koike

  • 3. Separation and detection of large phosphoproteins using Phos-tag™ SDS-PAGE, Nature Protocols, 4, 1513-1521 (2009), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike

Application using Phos-tag™ reagents
  • 1. Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling, Nat. Cell Biol., 9 ,1319-1326 (2007), C. I. Maeder et.al. M. A. Hink, A. Kinkhabwala, R. Mayr, P. I. H. Bastiaens and M. Knop

  • 2. Regulation of PKD by the MAPK p38d in Insulin Secretion and Glucose Homeostasis, Cell, 136, 235-248 (2009), G. Sumara, I. Formentini, S. Collins, I. Sumara, R. Windak, B. Bodenmiller, R. Ramracheya, D. Caille, H. Jiang, K. A. Platt, P. Meda, R. Aebersold, P. Rorsman, and R. Ricci1

  • 3. Dbf4-Dependent Cdc7 Kinase Links DNA Replication to the Segregation of Homologous Chromosomes in Meiosis I, Cell, 135, 662-678 (2008) ,J. Matos, J. J. Lipp, A. Bogdanova, S. Guillot, E. Okaz, M. Junqueira, A. Shevchenko, and W. Zachariae

  • 4. Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery, Cancer Res., 69, 5987-5995 (2009) , A. H. Sikkema, S. H. Diks, W. F.A. den Dunnen, A. ter Elst, F. J.G. Scherpen, E. W. Hoving, R. Ruijtenbeek, P. J. Boender, R. de Wijn, W. A. Kamps, M. P. Peppelenbosch, and E. S.J.M. de Bont

  • 5. Regulation of mitochondrial transport and inter-microtubule spacing by tau phosphorylation at the sites hyperphosphorylated in Alzheimer's disease, J. Neurosci.,32, 2430-2441 (2012), K.Shahpasand, I. Uemura, T.Saito, T.Asano, K.Hata, K.Shibata, Y.Toyoshima, M.Hasegawa, S.Hisanaga

  • 6. The Hsp90 Kinase Co-chaperone Cdc37 Regulates Tau Stability and Phoshorylation Dynamics, J. Biol. Chem., 286, 16976-16983 (2011) ., Umesh K. Jinwal, Justin H. Trotter, Jose F. Abisamobra, John Koren, III, Lisa Y. Lawson, Grant D. Vestal, John C. O’Leary, III, Amelia G. Johnson, Ying Jin, Jeffrey R. Jones, Qingyou Li, Edwin J. Weeber, and Chad A. Dickey

Tau proteins are used as the samples in Application 5 and 6.

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