用于通过 SDS-PAGE 分离磷酸化蛋白质

Phos-tag^® Acrylamide

仅需将Phos-tag^® Acrylamide与丙烯酰胺溶液混合并聚合，即可生成用于分离磷酸化和非磷酸化蛋白质的Phos-tag^® SDS-PAGE。

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。

◆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！

● 不论氨基酸残基的类型/位点如何，均可使用

可用于未知的磷酸化分析

可用于检测新的磷酸化位点（无需购买商品化磷酸化抗体、无需特意制备磷酸化抗体）

● 磷酸化位点数量/位点不同的磷酸形式也可分离

显示磷酸化程度和磷酸化形式

● 可同时检测磷酸化/非磷酸化形式

可定量各种磷酸化形式

可简单明确有无磷酸化

● 无需RI或特殊的仪器

实验成本低，操作简单。（SDS-PAGE的试剂→只要有设备即可使用）

● 电泳后可用于WB、MS分析，也可用于二维电泳

WB：可分析内源性蛋白

MS：可显示各磷酸化形式的磷酸化位点的组合

二维电泳：可分离相同等电点和分子量的磷酸化形式

适用于内源性蛋白的磷酸化分析！

◆Phos-tag^® SDS-PAGE的原理

Phos-tag^® Acrylamide的结构

1. 电泳中的磷酸化蛋白会被2个二价金属离子捕获

2. 磷酸化水平越高，电泳速度越慢

3. 根据磷酸化水平进行分离（即使磷酸化位点的数量相同，只要位置不同也可进行分离）

常规的SDS-PAGE（上图：非Phos-tag^® 条件下）中，无法确认底物是否被磷酸化。

◆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.

【使用示例：α-酪蛋白去磷酸化反应随时间的变化】

使用Phos-tag^® SDS-PAGE和常规SDS-PAGE分离碱性磷酸酶随时间（孵育时间：0~120 min）去磷酸化的α-酪蛋白样品。

◆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，对应指导手册请见相关资料。

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/48.html

已公开的验证蛋白列表，请点击

相关资料

Phos-tag 第6版说明书

说明书

【参考文献系列】Phos-tag™ SDS-PAGE

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

·  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