Phos-tag™ PAGE GUIDEBOOK

Application Data and References

Application Data 1

Impressions of users of Phos-tag™ Acrylamide and SuperSep Phos-tag™ precast gels are introduced herein. 'Ogawa provided comments and data of the sample, which had been assayed for kinase activity, separated by Phos-tag™ SDS-PAGE. Kimura provided the application data to a secondary electrophoresis, and Hosokawa provided the application data to the western blotting from Professor Tadayuki Ogawa of the University of Tokyo. In addition, we received data applied to two-dimensional electrophoretic migration from Professor Yayoi Kimura of Yokohama City University, as well as Western blotting applied data from Professor Sugiyama of Kochi University and Professor 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 between a phosphorylated protein and a non-phosphorylated protein by Phos-tag™ SDS-PAGE

Comparison between a phosphorylated protein and a non-phosphorylated protein by Phos-tag™ SDS-PAGE

In order to explore a kinase that phosphorylates about 40 kDa protein, the samples assayed for kinase activity were separated by Phos-tag™ SDS-PAGE. A lot of information was obtained from a small amount of samples as, compared to nonphosphorylated samples (NC), the proportion of phosphorylation/nonphosphorylation, degree of phosphorylation and distribution of population differed by the type of the kinase that reacted with other samples. With this information as the foothold, more detailed analyses using mass spectrometry, etc. were performed to identify the phosphorylation site specific to each kinase. (Reference : Ogawa T., Hirokawa N., Cell Rep., 2015 Sep. 22 ; 12(11) : 1774-88)

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

hnRNP K was separated by immune precipitation from a 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. Isoforms and modification sites of the various spots were then identified using a mass spectroscope.

2D electrophoretic migration

Different spots of phosphorylated states were detected at the same isoelectric point for each isoform!
(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:
    Professors Y. Kimura and H. Hirano, Biological Supramolecular Systems, Graduate School of Bionanosystems, Yokohama City University; and Professor O. Ohara, RCAI, Physics and Chemistry Research Institute.

Application Data 3
Determining fraction containing kinase for phosphorylating Dnmt1

Dnmt1: DNA methyltransferase

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:
    Professor Y. Sugiyama, and Professor 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:
    Professor T. Hosokawa, Memory Mechanisms Research Team, Circuit Function Mechanism Core, Brain Science Research Center, RIKEN; and Professor S. Hisanaga, Nerve Molecular Functions Research Room, Bioscience Studies, Institute of Science & Engineering, Tokyo Metropolitan University.

Application Data 5
Change in the phosphorylation level of MCL-1 under expression of wild-type/mutant Noxa

Wild-type (Wt) and mutant (3E, KR, 5A) Noxa genes were expressed in lung small cell carcinoma strain, H209 cells, and further separated into the cytoplasm (Cytosol) fraction and HM (heavy membrane, containing mitochondria in large quantities) fraction. MCL-1 (40 kDa) in these samples was isolated by Phos-tag™ SDS-PAGE and detected by Western blotting using anti-MCL-1 antibody.

Western

It was found that the phosphorylation level of MCL-1 increased in mitochondria in H209
cells expressed with wild-type and KR mutant Noxa.

【Papers giving the data】
Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.
Wataru Nakajima, Mark A. Hicks, Nobuyuki Tanaka, Geoffrey W. Krystal, and Hisashi Harada Cell Death and Disease (2014) 5, e1052; doi:10.1038/cddis.2014.6
Data provided by : Dr. Wataru Nakajima, Institute for Advanced Medical Sciences, Nippon Medical School.

Application Data 6
Time-course change in phosphorylation after X-ray irradiation of p53 and protein X

Human lung cancer-derived Lu99 cells were irradiated with X-ray (5 Gy), and the cells were retrieved in a time course. Cell extractions were prepared, and SDS-PAGE was performed using SuperSep Phos-tag™ (50 μmol/L), 10%, 13 wells. The gel was shaken in the transfer buffer containing 10 mM EDTA, and transferred to a PVDF membrane. The membrane was blocked with 2% Milk/TBS-T, and allowed to react with a primary antibody (upper row: p53, lower row: cell cycle-related protein, protein X). A chemiluminescent reagent was used for detection.

Western

It was shown that the accumulation of p53 reached a peak after 4 hours and phosphorylation of protein X changed with time as a result of X-ray irradiation.

Data provided by : Dr. Atsushi Enomoto, Center for Disease Biology and integrative Medicine, Faculty of Medicine, the University of Tokyo.

References

Regarding Phos-tag™ reagents:

  • 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate 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 Mn2+- Phos-tag™ SDS-PAGE

  • 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.

  • 7. PINK1-Phosphorylated Mitofusin 2 Is a Parkin Receptor for Culling Damaged Mitochondria, Science, Apr 2013; 340: 471 - 475., Yun Chen and Gerald W. Dorn, Ⅱ

  • 8. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice, Science, Dec 2015; 350: aad2459., Guohua Gong, Moshi Song, Gyorgy Csordas, Daniel P. Kelly, Scot J. Matkovich, and Gerald W. Dorn, Ⅱ

Zn2+-Phos-tag™ SDS-PAGE

  • 1. Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis, PLANT CELL, Feb 2013; 25: 535 - 544., Mátyás Medzihradszky, János Bindics, Éva Ádám, András Viczián, Éva Klement, Séverine Lorrain, Péter Gyula, Zsuzsanna Mérai, Christian

  • 2. MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast, Sci. Signal., Jan 2015; 8: ra5., Justin G. English, James P. Shellhammer, Michael Malahe, Patrick C. McCarter, Timothy C. Elston, and Henrik G. Dohlman

  • 3. Mechanism of Activity-Dependent Cargo Loading via the Phosphorylation of KIF3A by PKA and CaMKIIa., Neuron. 2015 Sep 2; 87(5):1022-35., Ichinose S, Ogawa T,and Hirokawa N.

  • 4. Microtubule Destabilizer KIF2A Undergoes Distinct Site-Specific Phosphorylation Cascades that Differentially Affect Neuronal Morphogenesis, Cell Reports, 2015 Sep 22; 12(11):1774-88

SuperSep™ Phos-tag™

  • 1. A laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag™ affinity electrophoresis. Int. J. Chem. 4, 1–8 (2012), Kinoshita-Kikuta, E., Kinoshita, E., and Koike, T.

  • 2. In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity, J. Cell Biol., Jul 2014; 206: 113 - 127., Sei Kuriyama, Eric Theveneau, Alexandre Benedetto, Maddy Parsons, Masamitsu Tanaka, Guillaume Charras, Alexandre Kabla, and Roberto Mayor

  • 3. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity, J. Cell Biol., Jan 2014; 204: 165 - 175., Maria M. Magiera, Elisabeth Gueydon, and Etienne Schwob

Increasing number of articles on Phos-tag™

Increasing number of articles on Phos-tag™