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Laboratory Chemicals > Product Information > P > Phos-tag™ series > Phos-tag™ Biotin

Western Blot Analysis of Phosphorylated proteins
- Chemiluminescent Detection using Biotinylated Phos-tag™ -

Phos-tag™ Biotin

Phos-tag™ Biotin is manufactured by Nard Institute.
Phos-tag™ BTL-104 and BTL-105 provide a sensitive method for detection of phosphorylated proteins on a PVDF membrane. This method needs streptavidin-conjugated horseradish (HRP) and chemiluminescent detection reagent.

INDEX
  1. Features
  2. Principle of phosphorylation of Affinity Chromatography
  3. Chemical Structures of Phos-tag™ Biotin
  4. Troubleshootings
  5. Product List
  6. References on Phos-tag™ Chemistry
  7. References on Phosphoproteome Analysis using Biotinylated Phos-tag™
Phos-tag™ Biotin


1. Features
  • Highly sensitive detection of phosphorylated proteins
  • The binding specificity of Phos-tag™ is independent on amino acid and sequence context.
  • Downstream procedures such as antibody reprobing and MS analysis are applicable.
  • The procedure is almost the same as that with an HRP-conjugated antibody.
2. Principle of Phos-tag™ Biotin on western blot analysis

Analysis image of western blot using Phos-tag™ Biotin

3. Chemical Structures of Phos-tag™ Biotin
Phos-tag™ Biotin


Phos-tag™ Biotin BTL-111
4. Troubleshootings
  1. Phos-tag™ BTL in Sol. E (10 μL) is large excess amount against Streptavidin-conjugated Horseradish Peroxidase in Sol. G (1 μL).  We obtained the same result using smaller amount of Phos-tag™ BTL (e.g., 1 mu;L Sol. E) and Sol. G (1 mu;L) used.  The user could adjust the volume of Sol. E to obtain the required sensitivity or save expenses.  If the volume of Sol. E is decreased, it is no need to the volume of the zinc(II) solution (Sol. F).  Little difference in the detection efficiency (due to the spacer effect) between BTL-104 and BTL-105 was observed in this protocol using the commercially available phosphorylated proteins and the Streptavidin-conjugated Horseradish Peroxidase (GE Healthcare Bio-Sciences).
  1. If the membrane is not thoroughly soaked with Sol. C, the background signal is high and spotted.  Furthermore, the protein signals are not observed (i.e., white spots in the right-side figure). Confirm that the membrane does not repel Sol. C.

  2. PVDF membrane is highly recommended for the Phos-tag™ BTL method.
troubleshooting 2

5. Product List

Description Manufacturer's code Package Size Wako Catalog No.
Phos-tag™ Biotin BTL-104 BTL-104 10 mg 301-93531
Phos-tag™ Biotin BTL-105 BTL-105 10 mg 308-93541
<Highly Sensitive type>
Phos-tag™ Biotin BTL-111   1 mM Aqueous Solution
BTL-111S1 0.1 mL
NEW
308-97201
   We recommend to try Phos-tag™ Biotin BTL-104 as your first choice due to the better solubility.
   Little difference in the detection efficiency (due to the spacer effect) between BTL-104 and BTL-105 was observed.
   Since BTL-111S1 has a long hydrophilic spacer, it has higher sensitivity than BTL-104.

6. References on Phos-tag™ Chemistry

  • 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 
  • Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex, Dalton Transactions, 1189-1193 (2004), E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro, and T. Koike
  • Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule, Journal of Lipid Research, 45, 2145-2150 (2004), T. Tanaka, H. Tsutsui, K. Hirano, T. Koike, A. Tokumura, and K. Satouchi
  • Production of 1,2-Didocosahexaenoyl Phosphatidylcholine by Bonito Muscle Lysophosphatidylcholine/Transacylase, Journal of Biochemistry, 136, 477-483 (2004), K. Hirano, H. Matsui, T. Tanaka, F. Matsuura, K. Satouchi, and T. Koike
  • Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins, Journal of Separation Science, 28, 155-162 (2005), E. Kinoshita, A. Yamada, H. Takeda, E. Kinoshita-Kikuta, and T. Koike
  • Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule, Analytical Chemistry, 77, 3979-3985 (2005), K. Inamori, M. Kyo, Y. Nishiya, Y. Inoue, T. Sonoda, E. Kinoshita, T. Koike, and Y. Katayama
  • 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
  • Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH, Proteomics, 6, 5088-5095 (2006), E. Kinoshita-Kikuta, E. Kinoshita, A. Yamada, M. Endo, and T. Koike
  • Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 360, 160-162 (2007), S. Yamada, H. Nakamura, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and Y. Shiro
  • Label-free kinase profiling using phosphate-affinity polyacrylamide gel electrophresis, Molecular & Cellular Proteomics, 6, 356-366 (2007), E. Kinoshita-Kikuta, Y. Aoki, E. Kinoshita, and T. Koike
  • A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike (The phosphate group at DNA-terminal is efficiently captured by Zn2+.Phos-tag.)
  • Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule Journal of Biomolecular Techniques, 18, 278-286 (2007), T. Nakanishi, E. Ando, M. Furuta, E. Kinoshita, E. Kikuta-Kinoshita, T. Koike, S. Tsunasawa, and O. Nishimura
  • A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE, Proteomics, 8, 2994-3003 (2008), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, S. Yamada, H. Nakamura, Y. Shiro, Y. Aoki, K. Okita, and T. Koike
  • FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway, Nature Structural & Molecular Biology, 15, 1138-1146 (2008), M. Ishiai, H. Kitao, A. Smogorzewska, J. Tomida, A. Kinomura, E. Uchida, A. Saberi, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, S. Tashiro, S. J. Elledge, and M. Takata
  • Two-dimensional phosphate affinity gel electrophoresis for the analysis of phosphoprotein isotypes, Electrophoresis, 30, 550-559 (2009), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, Y. Aoki, S. Ohie, Y. Mouri, and T. Koike
  • Formation of lysophosphatidic acid, a wound-healing lipid, during digestion of cabbage leaves, Bioscience, Biotechnology, and Biochemistry,73, 1293-300 (2009), T. Tanaka, G. Horiuchi, M. Matsuoka, K. Hirano, A. Tokumura, T. Koike, and K. Satouchi
  • A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides, Analytical Biochemistry, 388, 235-241, (2009), K. Takiyama, E. Kinoshita, E. Kinoshita-Kikuta, Y. Fujioka, Y. Kubo, and T. Koike
  • Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates, Analytical Biochemistry, 389, 83-85, (2009), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose, Proteomics, 9, 4098- 4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Ujihara, and T. Koike
  • 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
  • A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag, Rapid Communications in Mass Spectrometry, 24, 1075-1084 (2010), J. Morishige, M. Urikura, H. Takagi, K. Hirano, T. Koike, T. Tanaka, and K. Satouchi
  • Genotyping and mapping assay of single-nucleotide polymorphisms in CYP3A5 using DNA-binding zinc(II) complexes, Clinical Biochemistry, 43, 302-306 (2010), E. Kinoshita, E. Kinoshita-Kikuta, H. Nakashima, and T. Koike
  • The DNA-binding activity of mouse DNA methyltransferase 1 is ragulated phosphorylation with casein kinase 1σ/ε, Biochemical Journal, 427, 489-497 (2010), Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and I. Kameshita
  • Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling, Proteomics, 11, 319-323 (2011), E. Kinoshita and E. Kinoshita-Kikuta
  • A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the kinase reaction of a substrate peptide, Analytical Methods, 3, 1303-9 (2011), M. Somura, K. Takiyama, E. Kinoshita-Kikuta, E. Kinoshita and T. Koike
  • Highy sensitive detection of protein phosphorylation by using improved Phos-tag Biotin, Proteomics in press, E. Kinoshita, E. Kinoshita-Kikuta, Y. Sugiyama, Y. Fukuda, T. Ozeki and T. Koike

7. References on Phosphoproteome Analyses using Biotinylated Phos-tag™

  • Sept. 4, a Component of Presynaptic Scaffol and Lewy Bodies, Is Required for the Suppression of α-Synuclein Neurotoxicity, Neuron, 53, 519-533 (2007), M. Ihara, N. Yamasaki, A. Hagiwara, A. Tanigaki, A. Kitano, R. Hikawa, H. Tomimoto, M. Noda, M. Takanashi, H. Mori, N. Hattori, T. Miyakawa, and M. Kinoshita
  • Protein kinase Cδ binds TIRAP/Mal to participate in TLR signaling, Mol. Immunol., 44, 2257-2264 (2007), M. Kubo-Murai, K. Hazeki, N. Sukenobu, K. Yoshikawa, K. Nigorikawa, K. Inoue, T. Yamamoto, M. Matsumoto, T. Seya, N. Inoue, O. Hazeki
  • A Peptide Microarray for the Detection of Protein Kinase Activity in Cell Lysate, Anal. Sci ., 23, 271-275 (2007), S. Shigaki, T. Yamaji, X. Han, G. Yamanouchi, T. Sonoda, O. Okitsu, T. Mori, T. Niidome, and Y. Katayama
  • Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging, Anal. Biochem, 375, 223-231 (2008), T. Mori, K. Inamori, Y. Inoue, X. Han, G. Yamanouchi, T. Niidome, Y. Katayama
  • Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos, BMC Develop. Biol., 9, 21 (2009), X. Zhu, J. Dai, X. Tan, Y. Zhao, and W. Yang

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