Phos-tag™ Biotin

无特异性磷酸化抗体时的最佳选择！

　　Phos-tag™ Biotin是与生物素结合的Phos-tag™，可用于免疫印迹法检测磷酸化蛋白。Phos-tag™ Biotin BTL-104和BTL-105可灵敏检测PVDF膜上的磷酸化蛋白。

◆原理

◆优点、特色

●  无辐射。

●  无需PVDF膜的封闭处理。

●  Phos-tag™ 的特异性结合与氨基酸种类、序列无关。

●  可适用于免疫印迹和质谱分析等后续工作。

●   Phos-tag™ BTL母液可稳定保存至少6个月。

●  实验流程与使用HRP标记抗体相类似。

※BTL-104、BTL-105、BTL-111三者连接链（Linker）长度不一，但使用上基本相同。BTL-111灵敏度最高。

◆案例、应用

【使用例：在PVDF 膜上检测磷酸化蛋白】

　　转印在PVDF 膜上的磷酸化蛋白可精确检测到ng级水平，没有检测到相应的去磷酸化蛋白与非磷酸化蛋白的信号斑点。

　　免疫印迹检测磷酸化蛋白——Phos-tag ™ 生物素。

　　摘自Eiji Kinoshita ,et al., Mol.Cel.Proteomics (2006) 5: 749

【使用例：Phos-tag™ 生物素在检测蛋白激酶活性的微阵列（生物芯片）中的运用】

　　蛋白激酶是很多疾病诊断和药物筛选的靶标。近来有科研人员研发了一种检测胞内蛋白激酶活性的高灵敏度多肽微阵列。用微阵列点样机点样2nL体积的底物多肽溶液，使多肽固定在戊二醛预修饰的高氨基末端载玻片。

　　当多肽经细胞裂解液磷酸化后，用荧光标记的抗phosphotyrosine（磷酸化酪氨酸）抗体检测酪氨酸激酶，或者用Phos-tag™ 生物素，接着用荧光标记的亲和素检测丝氨酸或者苏氨酸激酶。之后用自动微阵列扫描仪检测荧光信号。多肽微阵列系统包括简单的多肽固定，只需少量样品，具有高密度阵列。重要的是，检测细胞裂解液蛋白激酶活性的灵敏度高。

　　因此多肽微阵列系统可用于高通量筛选细胞内激酶活性，可用于药物筛选和疾病诊断。

Phos-tag™ 系列

磷酸化蛋白新方法！

　　Phos-tag™是一种能与磷酸离子特异性结合的功能性分子。它可用于磷酸化蛋白的分离（Phos-tag™ Acrylamide）、Western Blot检测（Phos-tag™ Biotin）、蛋白纯化 （Phos-tag™Agarose）及质谱分析MALDI-TOF/MS （Phos-tag™ Mass Analytical Kit）。

◆Phos-tag™ 的基本结构：

特点：

与-2价磷酸根离子的亲和性和选择性高于其它阴离子

在pH 5-8的生理环境下生成稳定的复合物

◆原理

◆相关应用

◆相关产品

产品名称	用　　途
Phos-tag™ Acrylamide	分离: SDS – PAGE 分离不同磷酸化水平的蛋白
SuperSep Phos-tag™	分离: 预制胶中含有50μM Phos-tag™ Acrylamide
Phos-tag™ Biotin	检测: 代替 Western Blot 检测中的磷酸化抗体
Phos-tag™ Agarose	纯化: 通用柱层析，纯化磷酸化蛋白
Phos-tag™ Mass 　Analytical Kit	分析: 用于质谱 MALDI-TOF/MS 分析，提高磷酸化分子的检测灵敏度

phos-tag™由日本广岛大学研究生院医齿药学综合研究科医药分子功能科学研究室开发。

更多产品信息，请点击：http://phos-tag.jp

Phos-tag 第5版说明书	Phos-tag系列 ver 5
Phos-tag生物素操作手册	说明书

Q： BTL-104和BTL-111的区别？

A： BTL-104的溶解度更高，BTL-111的灵敏度更高。

 

Q： 检测灵敏度达到什么水平？

A： 可达到ng级别。需要使用化学发光试剂，比如ImmunoStar LD(Wako)。

 

Q： 使用该产品还需要别的试剂或则耗材吗？

A： 硝酸锌（Zn(NO₃)₂）溶液和亲和素标记的HRP（(GE Healthcare Bio-Sciences:       RPN1231)。制备Phos-tag™生物素与亲和素标记的HRP偶联物时需要使用离心过  滤装置(NMWL = 30,000, Nanosep™ 30K, Pall Life Sciences).

 

Q： Phos-tag^™生物素使用的次数？

A： 主要决定于使用次数和使用量，以下实验次数仅作参考：

     BTL-104：130~1300次；

      BTL-111 1mM溶液：10~100次。

 

Q： 可测定磷酸化蛋白？

A： 根据条带的浓度，可进行半定量分析。

 

Q： 能够确定结合磷酸化基团的数目？

A： 不能。

Q： 能否剥除（strip）Phos-tag™生物素？

A： 可以。与含有62.5mM Tris-HCl（pH6.8），2%（w/v）SDS和0.1M 2-巯基乙醇溶液  混合后，震荡15分钟。用1×TBS-T洗涤3次，每次10分钟。

 

Q： 推荐使用哪种膜？

A： 建议使用PVDF膜。

 

Q： 使用Phos-tag™ 生物素是否需要封闭？

A： 不需要。封闭会降低检测灵敏度。

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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

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• 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