◆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.

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

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