Phos-tag™ Mass Analytical Kit

用于 MALDI-TOF/MS检测，提高检测灵敏度！

　　用于质谱分析的试剂套装。

　　Phos-tag Mass Analytical Kit 是用于质谱分析的试剂套装，可配套 MALDI-TOF/MS 使用。可检测磷酸化分子- Phos-tag^® 复合物，通常可提高低磷酸化分子的检测灵敏度。

试剂盒内容：

● Phos-tag™ MS-101L  5 mg（［C27H29N6O64Zn2］3+ MW：581.4）

● Phos-tag™ MS-101H 5 mg（［C27H29N6O68Zn2］3+ MW：589.4）

● Phos-tag™ MS-101N 10 mg（［C27H29N6OZn2］3+ MW：584.3）

◆原理：

◆优点、特色：

● CH₃COO- 等价结合在 Phos-tag™ MS-101 上。

● 在溶液中，不含有阴离子的 Phos-tag™ MS-101 带有＋3价。

● 检测前需制备 1 mM 的 Phos-tag™ MS-101L，MS-101H 或者 MS-101N（溶于水）。

◆案例、应用：

【使用例子：检测 Phos-tag™ – 磷酸化 LPA 复合体】

由于正电荷增大磷酸化 LPA 检测灵敏度上升

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：

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6版说明书

Phos-tag系列 ver. 8

Q.     Phos-tag™ Mass 用于实验可以使用多少次？

A.     如果每次用量为 5 μL，至少可以使用 1000 次。

Q.     如何选择使用 Phos-tag™ MS-101L，Phos-tag™ MS-101H 和 Phos-tag™ MS-101N ？

A.     Phos-tag™ 101N 含有自然存在的 Zn，101L 与 101H 分别含有 Zn 的同位素 ⁶⁴Zn 和 ⁶⁸Zn。

　    请参考以下建议：

　    摸索条件时使用 101N，其中含有多种同位素，结果比较详细；

　    鉴定磷酸基团是否存在，使用 101L 和 101H，这些试剂分别包含 ⁶⁴Zn 和 ⁶⁸Zn。使用这些试剂检测同一个样品时会产生不同的荷质比。

Q.     如果想测定经过 Phos-tag™ SDS-PAGE 分离得到的样品，是否必须要在凝胶消化之前去除 Phos-tag™？

A.     没有必要。SDS-PAGE 结束之后根据一般的凝胶消化方法进行操作即可。

Q.     能否用于 ESI 质谱？

A.     是的，可以使用。请参考下面的文献，这篇报道使用 Phos-tag™ MS-101N 进行 ESI-MS 分析。

　     在实验过程中，使用了中性溶液，若为酸性溶液会导致 Phos-tag ™ 分离。

        【参考文献】 Anal. Chem. (2008), 80, 2531-2538 (MS-101N ESI-MS)

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·  Advances in crop proteomics: PTMs of proteins under abiotic stress[J]. Proteomics, 2016, 16(5): 847-865，Wu X, Gong F, Cao D, et al.

·  Cyclin-Dependent Kinase Co-Ordinates Carbohydrate Metabolism and Cell Cycle in S. cerevisiae[J]. Molecular cell, 2016, 62(4): 546-557，Zhao G, Chen Y, Carey L, et al.

·  Carbon Monoxide Gas Is Not Inert, but Global, in Its Consequences for Bacterial Gene Expression, Iron Acquisition, and Antibiotic Resistance[J]. Antioxidants & redox signaling, 2016，Wareham L K, Begg R, Jesse H E, et al.

·  Two-layer regulation of PAQR3 on ATG14-linked class III PtdIns3K activation upon glucose starvation[J]. Autophagy, 2016: 1-2，Xu D, Wang Z, Chen Y.

·  Regulation of sphingolipid biosynthesis by the morphogenesis checkpoint kinase Swe1[J]. Journal of Biological Chemistry, 2016, 291(5): 2524-2534，Chauhan N, Han G, Somashekarappa N, et al.

·  PAX5 tyrosine phosphorylation by SYK co-operatively functions with its serine phosphorylation to cancel the PAX5-dependent repression of BLIMP1: A mechanism for antigen-triggered plasma cell differentiation[J]. Biochemical and biophysical research communications, 2016, 475(2): 176-181，Inagaki Y, Hayakawa F, Hirano D, et al.

·  A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock[J]. Journal of Bacteriology, 2016: JB. 00235-16，Boyd J S, Cheng R R, Paddock M L, et al.

·  HuR mediates motility of human bone marrow-derived mesenchymal stem cells triggered by sphingosine 1-phosphate in liver fibrosis[J]. Journal of Molecular Medicine, 2016: 1-14，Chang N, Ge J, Xiu L, et al.

·  Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts[J]. Biochemistry and Biophysics Reports, 2016，Kitakaze K, Tasaki C, Tajima Y, et al.

·  Roseotoxin B Improves Allergic Contact Dermatitis through a Unique Anti-inflammatory Mechanism Involving Excessive Activation of Autophagy in Activated T-Lymphocytes[J]. Journal of Investigative Dermatology, 2016，Wang X, Hu C, Wu X, et al.

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

  
  

蛋白磷酸化研究的预制胶

SuperSep Phos-tag™

　　SuperSep Phos-tag™ 是研究蛋白磷酸化的方法，无需特异性磷酸化抗体或者同位素标记。

◆SuperSep Phos-tag™

　　Phos-tag™ 是一种预制胶，预先加入了 50 μmol/L 的 Phostag™ Acrylamide，打开包装即可直接使用。预制胶中含有锌作为金属离子，在中心凝胶缓冲液中保存稳定性很好，得到的带条结果也很整齐。

　　磷酸化蛋白和非磷酸化蛋白作为不同条带分离。

　　分离后，胶可用于考马斯亮蓝染色，免疫印迹和质谱实验。

已公开的验证蛋白列表，请点击

◆Phos-tag™ SDS-PAGE 的原理

◆在 HighWire Search 上搜索到的论文数

◆运用

利用 p35 的丙氨酸突变体确定 Cdk5 激活 p35 的磷酸化位点

　　p35 常见的磷酸化位点是 Ser8 和 Thr138。但是 Ser8 和 Thr138 位点往往会发生丙氨酸突变，产生３种突变体（Ser8 突变体：S8A，Thr138 突变体：T138A，Ser8 和 Thr138 双突变体：2A）。这３种突变体、野生型 p35、Cdk5 和没有激酶活性的 Cdk5 都来源于 COS-7 细胞。这些细胞裂解液用Phos-tag™ SDS-PAGE 和 Western blotting 进行检测（检测抗体：p35 抗体）。

100 μM Phos-tag ™ 丙烯酰胺, 7.5% 聚丙烯酰胺凝胶

可明确磷酸化位点和条带迁移率的关系！

– 泳道１（条带 L2 和 L4）和泳道５（条带 M1）：p35 在 Cdk5 的作用下发生了磷酸化；

– 泳道１（条带 L2 和 L4）和泳道３（条带 L2 和 L4）：在无激酶活性 Cdk5 的作用下，大约有一半 p35 蛋白在 Thr138 位点发生磷酸化，同样在 138 位发生突变的 p35 蛋白亦是如此。

– 泳道５ （条带 M1）和泳道６（条带 L3 和 L4）：Ser8 和 Thr138 是主要的磷酸化位点；

– 泳道５（条带 M1）、泳道７（条带L1和L2）和泳道８（条带M2）：条带 M1 是 Ser8 和T hr138 都发生磷酸化的条带；

条带 M2 是只有 Ser8 磷酸化的条带；条带 L1 和 L2 是只有 Thr138 磷酸化的条带。

※ 条带 L1 和 L3 中的Ｘ是不确定哪个位点发生磷酸化的条带；

※ 条带L4是非磷酸化的 p35 。

【参考文献】

▪ 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.

【结果提供】

理化学研究所 脑科学综合研究中心 回路功能研究核心 记忆功能研究团队 细川智永（Dr. T. Hosokawa）

首都大学东京 理工学研究科 生命科学专业 神经分子功能研究室 久永真市（Dr. S. Hisanaga）

检测含有 Dnmt1 磷酸化激酶的片段

我们可以确定在片段中含有目的激酶！

① 采用亲和色谱法从鼠脑提取液中纯化 GST-Dnmt1（1-290）结合蛋白

② 使用 0.3 M 和 1 M NaCl 的 DNA 纤维素柱洗脱得到目的蛋白

③ GST-Dnmt1（1-290）作为体外激酶实验的反应底物

④ Phos-tag ™ SDS-PAGE 用于Western blotting，确定迁移条带中每个片段的激酶活性

【参考文献】

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.

【结果提供】

高知大学 综合研究中心 生命、功能物质部门 实验实习机器设施 杉山康宪（Dr. Y. Sugiyama）

香川大学 农学部 应用生物科学科 动物功能生化学研究室 龟下勇（Dr. I. Kameshita）

二维电泳中的应用：分析 hnRNP K 磷酸化异构体

　　小鼠巨噬细胞 J774.1 经 LPS 刺激后，裂解细胞，经过免疫沉淀法分离得到 hnRNP K。在二维电泳中，一维是 IPG 胶，二维是 Phos-tag ™ SDS-PAGE，可分离 hnRNP K 的异构体。利用质谱仪，可以确认不同的点代表不同的亚型或修饰蛋白。

同一个等电点的位置上，不同位点发生磷酸化都可以被区分开来

（例: spots 6 vs. 8 and spots 4 vs. 7）

【参考文献】

▪ 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.

【结果提供】

横滨市立大学 生命纳米系统科学研究科 生物体超分子系统科学专业 木村弥生（Dr. Y. Kimura）、平野久（Dr. H. Hirano）

理化学研究所 RCAI 小原收

◆备注

样品制备：

Phos-tag SDS-PAGE 对于蛋白样品中的杂质非常敏感，尤其是螯合剂，钒酸，无机盐，表面活性剂这类物质。

强烈建议在 Phos-tag SDS-PAGE 之前通过 TCA 沉淀或渗析法降低杂质含量。

转膜前处理：

另一个必须的步骤是在转膜前，用 EDTA 去除凝胶中的金属离子（Mn²⁺ 或者Zn²⁺）；该步骤可提高蛋白的转膜效率。

●　分别准备 10 mmol/L 含 EDTA 和不含 EDTA 两种 1x transfer buffer。

●　将凝胶浸泡在含 10 mmol/L EDTA 的 1x transfer buffer，至少 20 分钟，温和摇晃。更换新缓冲液，重复３次。

●　将凝胶浸泡在不含 10 mmol/L EDTA 的 1x transfer buffer，10 分钟，温和摇晃。

●　转膜操作*。

* 建议用湿法转膜，以提高转膜效率。

◆质量控制

　　每一批 SuperSep Phos-tag™，出厂前均根据其产品规格进行测试，以保证可分离磷酸化和非磷酸化蛋白，以及他们的分离成都在正常参数内。

◆保存温度

2-10℃

◆产品信息

用于 Bio-Rad 伯乐电泳仪

用于 Life Technologies 电泳仪

用于 Wako EasySeperator 电泳仪

◆相关产品

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：

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：

Phos-tag 第6版说明书

Phos-tag系列 ver. 8

参考文献

Phos-tag™ Biotin

无特异性磷酸化抗体时的理想选择！

　　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 灵敏度更高。

◆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 2 nL 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

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：

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 1 mM 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 62.5 mM 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。

【参考文献】

Phos-tag™ Acrylamide

SDS-PAGE 分离不同磷酸化水平的蛋白！

　　在不使用放射性同位素的情况下，利用 Phos-tag™ SDS-PAGE 即可分离不同条带中的磷酸化和非磷酸化蛋白。分离后的凝胶可用于 Western blotting 和质谱分析等后续实验。

　　Phos-tag™ SDS-PAGE 操作简单，只需在常规 SDS-PAGE 胶中加入 Phos-tag™ Acrylamide 和 Mn²⁺ 或者 Zn²⁺ 即可进行实验。在电泳过程中，磷酸化蛋白的磷酸基团与 Phos-tag™ 中的二价金属离子相结合，降低其迁移速度，从而可区分磷酸化与非磷酸化蛋白。

◆原理

◆优点、特色

●  采用 Phos-tag™ SDS-PAGE 可轻松分离磷酸化蛋白

　  无任何放射性元素及化学标记！

●  可检测不同磷酸化水平的磷酸化蛋白

　  无需任何磷酸化抗体！

●  适用于内源性蛋白的磷酸化分析！

◆案例、应用

【使用Phos-tag™ SDS-PAGE的磷酸化/非磷酸化蛋白比较】

我推荐使用Phos-tag ™ ——东京大学研究院医学研究科 小川觉之

　　Phos-tag ™ 是专为研究磷酸化蛋白而新开发出来的试剂。此产品使用方便，不但可用于体外实验，还能定量分析体内蛋白的磷酸化水平。Phos-tag ™ SDS-PAGE 可用于常规电泳实验，无需购买特殊设备，性价比高。传统蛋白磷酸化的研究需要特异的磷酸化抗体、RI 等其它试剂，操作复杂，花费大，且放射性元素会有安全隐患，而 Phos-tag ™ 的出现恰恰可以弥补这些缺点，为磷酸化蛋白研究提供新的方向。

磷酸化蛋白和非磷酸化蛋白利用Phos-tag ™ SDS-PAGE 的分离效果图

　　Lane 1 为非磷酸化蛋白，Lane 2-5 为磷酸化蛋白，各蛋白因磷酸化状态不同而条带迁移率也有所不同。

　　磷酸化/ 非磷酸化蛋白的数量比、磷酸化程度、磷酸化蛋白的丰度等都可根据条带迁移和条带浓度求得。

【资料提供】

日本东京大学研究生院医学系研究科

【二维电泳中的应用：分析 hnRNP K 磷酸化异构体】

　　小鼠巨噬细胞 J774.1 经 LPS 刺激后，裂解细胞，经过免疫沉淀法分离得到 hnRNP K 。在二维电泳中，一维是IPG 胶，二维是 Phos-tag ™ SDS-PAGE，可分离 hnRNP K 的异构体。利用质谱仪，可以确认不同的点代表不同的亚型或修饰蛋白。

二维电泳

　　同一个等电点的位置上，不同位点发生磷酸化都可以被区分开来（例: spots 6 vs. 8 and spots 4 vs. 7）

【参考文献】

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.

【结果提供】

　　横滨市立大学 生命纳米系统科学研究科 生物体超分子系统科学专业 木村弥生（Dr. Y. Kimura）、平野久（Dr. H. Hirano）理化学研究所 RCAI 小原收

【EGF 刺激前后MAPK 磷酸化水平的变化】

　　常规 SDS-PAGE 和Phos-tag^TM SDS-PAGE 后迚行克疫印迹实验分析 EGF 刺激的 A431 细胞中 MAPK 磷酸化水平。

摘自 Kinoshita-Kikuta, E. et al., Mol.Cell. Proteomics. (2007)6: 356.

操作视频，请点击：

样品处理（TCA沉淀）：http://labchem.fujifilm-wako.com.cn/resources/show/47.html

凝胶制备：http://labchem.fujifilm-wako.com.cn/resources/show/47.html

已公开的验证蛋白列表，请点击

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

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：

Phos-tag 第6版说明书

说明书

1.     Phos-tag^® Acrylamide的溶解

5 mmmol/ Phos-tag^® 液体 (3v/v% 甲醇）：

1） 10 mg  Phos-tag^® Acrylamide 里加入 0.1 mL 甲醇

2） 使用枪头搅拌混合直至完全溶解。

3） 加 3.2 mL 蒸馏水， 用枪头混匀。

      2-8℃ 避光保存。不适合零度以下保存。建议保存时间６个月。

      注意：避免溶解过程出现白色悬浮颗粒。

2.     α-Casein, from Bovine Milk, Dephosphorylated（038-23221），阳性对照（含有磷酸化和非磷酸化α-Casein），如何使用？

　　用水或者上样 buffer 溶解。用水溶解后，冷冻保存。电泳条件：Phos-tag^® 50 umol/L,分离胶浓度 10% 。

　　电流：30 mM，1小时。

3.     用Alkaline Phosphatase(for Biochemistry)（018-10693）进行磷酸化蛋白的去磷酸化反应体系。

　　 37℃，过夜。# 10 mg/mL phosphorylated protein 50 μL
　　 # 0.50 M Tris/HCl buffer (pH 9.0) containing 0.10 M MgCl2 10 μL
　　 # Sterilized water 39 μL
　　 # Alkaline phosphatase（018-10693）. 0.3 unit / 1 μL 有一点需要注意：ALP 活性化使用 Mg 离子，相同的非磷酸化蛋白质用 ALP

           处理的样品的条带和没有用ALP处理的样品的条带的位置不同。

4.     Phos-tag^® SDS-PAGE 实验没有成功分离磷酸化蛋白：

　 1） 使用 α-Casein, from Bovine Milk, Dephosphorylated（038-23221）作为阳性对照，确认实验条件和试剂均没有问题。

　 2） 可使用 Phos-tag^® Biotin 检测样品中是否有磷酸化蛋白。确认有磷酸化蛋白后，再通过 Phos-tag^® SDS-PAGE 进行分离鉴定。

　 3） 经质谱鉴定有表达磷酸化蛋白，建议增大样品的含量，可使用 Phos-tag^® Agarose 进行磷酸化蛋白的富集。磷酸化蛋白含量过低，

           会影响其分离效果。

　 4） 文献报道有表达磷酸化蛋白，或者同源蛋白有表达磷酸化蛋白的，建议用 Phos-tag^® Biotin 先确认样品中是否有磷酸化蛋白。

　 5） 建议样品的 pH 值在７左右。酸性或者碱性条件下，Mn²⁺ -Phos-tag^® 与磷酸化基团的特异性结合较差。

　 6） 避免样品中含有高浓度的还原剂，变性剂，表面活性剂等。β-巯基乙醇浓度不高于 0.2 M（或者５%）。

　 7） 进行 Phos-tag^® SDS-PAGE 的最佳样品是纯化的蛋白。如果是细胞裂解液，体外激酶反应液，组织均浆液等，需要摸索最佳的分

           离胶，Phos-tag^® Acylamide 的浓度。建议 Phos-tag^® Acrylamide 浓度从 50 μM 开始摸索。

5.     Phos-tag^® SDS-PAGE 凝胶用于 Western Blotting 实验的优化建议：

1） 可以检测的样品包括体外激酶反应体系，细胞裂解液，组织均浆液。

2） 每孔样品的上样量是 10~30 μg（请根据蛋白表达量进行调整）

3） 制备样品中含有的还原剂、变性剂、螯合剂、钒酸等会使电泳条带发生弯曲或者拖尾。通过 TCA 沉淀或渗析法降低杂质含量。

4） 建议样品的 pH 值在７左右。如果加入上样缓冲液后溶液显黄色或者橙色，加入 Tris 缓冲液调整 pH 值为７。

5） 目的蛋白分子量大于 60 kDa，分离胶的丙烯酰胺浓度为６%；目的蛋白分子量小于 60 kDa，分离胶的丙烯酰胺浓度为８%。

6） 如果样品中含有大量蛋白，比如细胞裂解液，组织均浆液，Phos-tag^® Acylamide 浓度为 5~25 uM。

　　若目的蛋白浓度低，建议 Phos-tag^® Acylamide 浓度为 100 uM。

7） Phos-tag^® SDS-PAGE凝胶用于 Western Blotting 实验，湿法转膜建议：10 mM EDTA 的转移缓冲液处理凝胶 10 min，不含有

      EDTA 的转移缓冲液处理凝胶 10 min。重复３次。强烈建议湿法转膜。

8） Phos-tag^® SDS PAGE 半干法转膜建议：

  i.　 电泳后用含有 EDTA 的转移缓冲液处理凝胶，EDTA的浓度为 100 mM。100 mM EDTA 的转移缓冲液处理凝胶 10 min，不含有

        EDTA的转移缓冲液处理凝胶 10 min。重复３次。

  ii.    转膜的电流值提高２%~３%, 延长时间２成。

  iii.   转膜的缓冲液加 SDS，加到大约 0.05~0.2%，转膜效率会提高。

  9） 使用目的蛋白的非磷酸化抗体即可。比如检测各种肿瘤细胞系中 Src 激酶活性实验，用 Src 的非磷酸化抗体即可。

10） 富士胶片和光的 WIDE-VIEW^™ Prestained Protein Siza MarkerIII（230-02461）可检测作为转膜效率，但是无法判断分子量。

11） 一般预染的蛋白 marker 在 Phos-tag^® SDS-PAGE 中条带会弯曲，无法判断蛋白分子量。

12） 不能确认磷酸化蛋白和非磷酸化蛋白的分离，请进行常规的 SDS-PAGE，Western Blotting 实验。比对目的蛋白的迁移率。

13） 不能确认是因为蛋白发生磷酸化还是出现降解造成蛋白条带迁移，请进行常规的 SDS-PAGE 实验，确认不会出现条带迁移。

14） 目的蛋白磷酸化与非磷酸化分离效果不佳，使用 α-Casein、from Bovine Milk、Dephosphorylated（038-23221）作为阳性对照，

         确认实验条件和试剂均没有问题。如果确认能够分离，调整分离胶，Phos-tag^® Acylamide 的浓度。建议使用品质佳的 MnCl₂

       （139-00722）。

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 References on Phos-tag™ Chemistry

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·  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

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