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-tagTM 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://www.boppard.cn/resources/show/47.html

凝胶制备：http://www.boppard.cn/resources/show/48.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

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 系列产品 Ver.7 2

说明书

1.     Phos-tag® Acrylamide的溶解

5mmmol/ Phos-tag® 液体 (3v/v% 甲醇）：

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

2)    使用枪头搅拌混合直至完全溶解。

3)     加3.2mL 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® 50umol/L,分离胶浓度 10%。

　　 电流：30mM，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0.2M（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50uM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10~30ug（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60kDa，分离胶的丙烯酰胺浓度为6%；目的蛋白分子量小于60kDa,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5~25uM。

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

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

　　处理凝胶10min，不含有EDTA的转移缓冲液处理凝胶10min。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 100mM。100mM EDTA的转移

　　　　　缓冲液处理凝胶10min，不含有EDTA的转移缓冲液处理凝胶10min。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） 和光的WIDE-VIEW^TM 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

·  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

 

Phos-tag™ Agarose

亲和层析纯化磷酸化蛋白

　　填入色谱柱中使用。可分离、纯化、浓缩磷酸化蛋白。不使用界面活性剂、还原剂，可得到状态近似生物体内的磷酸化蛋白。

◆原理：

◆优点、特色：

●　缓冲液不含有界面活性剂、还原剂。

●　与亲和层析方法类似。

●　可在1小时内纯化磷酸化蛋白。

●　Phos-tag™ Agarose捕获结合到Tyr、Thr、Ser、Asp、His等氨基酸、糖类、脂类上的无机磷酸根和大

　  量二价磷酸根。

●　可在生理条件下（pH7.5）捕捉蛋白。

●　纯化后的产物可用于Co-IP实验和其他蛋白活性实验。

◆案例、应用：

【使用例子：A431裂解液中的磷酸化蛋白的纯化】

　　把Phos-tag™ 填充到柱里，再加上A431 裂解液。

　　SYPRO Ruby染色（左图）再使用Anti-p Tyr抗体进行免疫印迹（右图），检测出结果。

　　结果确认磷酸化蛋白浓缩在柱吸附层里。

　　M：分子量标记

　　Lane 1：未吸附层

　　Lane 2：吸附层

　　Lane 3：柱清洗层

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

说明书

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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

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