产品说明书：

AAL-107.pdf

Phos-tag^TM SDS-PAGE 实验指导手册：

Phos-tag SDS-PAGE 实验指导手册.pdf

磷酸化系列产品：

磷酸化系列产品ver.7.pdf

产品相关FAQ：

AAL-107  FAQ.pdf

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！

·相关应用

·案例

【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，Lane2-5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™ SDS-PAGE 后进行免疫印迹实验分析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://yjinbio.sysweb6543.7cheng24.com/downloaddetail_14719.html

已公开的验证蛋白列表，请点击：

·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。

   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）.

相关验证蛋白：

更多产品信息，请点击：http://phos-tag.jp

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