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。

◆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Thr138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.,

【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。

◆产品信息

用于Bio-Rad伯乐电泳仪

货号	品名	电泳仪	规格
198-17981	SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 83×100×3.9mm	Mini-PROTEAN®  Tetra Cell (Bio-Rad Laboratories, Inc.)	5 块
195-17991	SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 83×100×3.9mm		5 块

用于Life Technologies电泳仪

货号	品名	电泳仪	规格
192-18001	SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 100×100×6.6mm	XCell SureLock®  Mini-Cell (Life Technologies, Inc.)	5 块
199-18011	SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 100×100×6.6mm		5 块

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 ver 5

Q1.	我们可以采用哪种凝胶染色法？
A1.	所有的染色法都可使用，最常用的例如考马斯亮蓝染色法，负染，银染和荧光染色等。
Q2.	用考马斯亮蓝染法染色，着色不明显。
A2.	在微波炉内进行脱色，会取得比较满意的效果。 方法：将染色的胶放在100毫升去离子水里，用擦拭纸包裹胶，再放进微波炉加热几分钟后更换去离子水，并重复上述步骤3或4次。请注意防止盛放胶的容器温度过高。
Q3.	此款产品能否用于免疫印迹？
A3.	可以，如果用EDTA清除胶里面含有的锌，可以提高转膜的效率。 方法：胶浸在含有10 mmol/L EDTA的转移缓冲液（25 mmol/L tris、192   mmol/L甘氨酸，10％甲醇）里轻轻搅拌10分钟。重复上述步骤3次。然后放进不含EDTA的转移缓冲液（25 mmol/L tris、192   mmol/L的甘氨酸，10％甲醇）里搅拌10分钟并转移到PVDF膜或NC膜（硝酸纤维素膜）上。
Q4.	条带扭曲了。
A4.	含有EDTA，无机盐，表面活性剂等的样品可能会导致条带弯曲或者拖尾。通过TCA或透析沉淀脱盐样品。空白泳道也会导致相邻样品条带弯曲，在空白泳道加与样品相同体积的样品缓冲液(x1)。
Q5.	磷酸化蛋白和非磷酸化蛋白不能分离。
A5.	将β–酪蛋白（038-23221）作为Phos-tag SDS-PAGE电泳的阳性对照，将用碱性磷酸酶处理的β–酪蛋白作为阴性对照，并检查条带的迁移。如果只有一个条带，可能是由于Phos-tag™或丙烯酰胺的浓度没有优化导致磷酸化和非磷酸化蛋白不能分离。
Q6.	可用在细胞的粗提物上吗？
A6.	可以的，可能Rf值可能有稍低，由于目的蛋白的因素，条带可能会比较模糊。
Q7.	上样量多少？
A7.	1至5微克纯化的蛋白（考马斯亮蓝染法），10至30微克的组织或细胞提取物（取决于蛋白质的表达量）。 *这是推荐的用量，可以先进行常规的SDS-PAGE和免疫印迹法，确定合适的上样量。
Q8.	使用哪种蛋白marker？
A8.	不推荐使用蛋白marker。该款产品不需要蛋白marker。因此，建议用来源于大肠杆菌的重组蛋白或者是非磷酸化样品作为阴性对照代替蛋白marker。
Q9.	此款产品的配离子是什么？
A9.	锌离子
Q10.	我们怎么知道是因为蛋白发生磷酸化条带才会发生迁移？
A10.	使用12.5% SuperSep™   Ace(Wako 目录No. 199-14971)进行电泳（有相同的胶浓度），检查目标蛋白质是否降解。

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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