Myxochelin A

　　人强效5-脂氧合酶抑制剂（5-LO），该酶催化花生四烯酸转化成白三烯的前两部，是众所周知的炎症和过敏反应介质。

铁离子螯合物

　　抗癌症抗生素，在体外抑制肿瘤细胞侵袭。

抗菌化合物

　　具有清除自由基活性的抗氧化剂。

参考文献

抗硫酸角质素单克隆抗体（R-10G）

人iPS/ES细胞标记抗体

◆特点

●　高选择性识别人iPS/ ES细胞，几乎不识别EC细胞

●　使用简便的IgG₁型抗体

◆案例

〈检测条件〉 一抗 :    A. TRA-1-81抗体（阳性对照）    B. 抗硫酸角质素，单克隆抗体（R-10G）   二抗 :    FITC标记二抗   细胞：人iPS细胞 201B7株 On-Feeder培养 蓝 : DAPI 绿 : FITC（抗体）

参考文献

Kawabe, K. et al., Glycobiology, 23(3), 322 (2013).

欲了解更多相关产品请点击文字：A-83-01, MF

Tau抗体系列

用于研究老年痴呆症

　　Tau属于微管结合蛋白，主要在中枢神经系统中神经细胞表达，控制微管的稳定化。在患有阿尔茨海默氏病的患者脑中，磷酸化Tau蛋白异常聚集形成神经原纤维缠结，据报道缠结数量与老年痴呆症的严重程度相关联。因此，为探究阿尔茨海默氏病的病因以及开发治疗药物，对Tau进行积极研究。

　　Tau是含有六种同源异构体以及超过40个磷酸化位点，通过同类分子聚合可得到低聚物，颗粒凝集体，纤维等不同形态。

　　现在，推出了4种Tau抗体（3R-Tau抗体、4R-Tau抗体、磷酸化Tau T181抗体、磷酸化Tau S199抗体）供客户选择。

◆特点

　　●　价格优惠

　　●　高性能单克隆抗体

　　●　根据不同的识别对象推出4种抗体

◆抗体概要

数据提供

　3R-Tau/4R-Tau抗体：名古屋大学大学院医学系神经科学研究生院 石垣先生、藤岡先生
　磷酸化Tau T181/Tau S199抗体：同志社大学生命科学部医工学系统神经病理学 宮坂先生

◆性能

相关资料

◆产品资料

Tau抗体系列

◆相关产品列表

动物样本的氨检测用

LabAssay™ Ammonia

　　氨主要产生于肠道和肾脏。在生物体内产生的氨是利用肝脏中的尿素循环转变成尿素，然后从尿液中排出。

　　本品是用亚硝基铁氰化钠作为催化剂，以利用苯酚、次氯酸的贝特洛反应的比色法为基础的氨检测试剂。

　　LabAssay™ 系列是研究用试剂。不可用于诊断。

◆特点

●　只需少量样本即可进行检测

●　可使用微孔板检测多个样本

●　可检测小鼠等动物样本

◆检测原理

　　氨和苯酚在催化剂亚硝基铁氰化钠的作用下置换生成二氧二苯胺。在强碱条件下，利用与次氯酸钠反应生成靛酚。通过测定靛酚呈现出蓝色的吸光度，从而测定出样本中的氮氨浓度。

◆试剂盒组成

●　除蛋白试剂——100 mL×1瓶

●　发色剂A——50 mL×1瓶

●　发色剂B——25 mL×1瓶

●　发色剂C——50 mL×1瓶

●　氨标准溶液——15 mL×1瓶

●　标准溶液用稀释液——20 mL×1瓶

◆性能

●　标准曲线范围——100~400 μg/dL（μg/100 mL）

●　检测时间——约70分钟

●　样本量——70 μL

●　检测波长——630 nm

〈标准曲线〉

◆产品列表

◆相关产品

 皮卡丘素抗体

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

用于Life Technologies电泳仪

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：

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

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·  Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation[J]. Nature communications, 2016, 7，Mitterer V, Murat G, Réty S, et al.

·  Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors[J]. Biochemical Journal, 2016: BCJ20160557，Ito G, Katsemonova K, Tonelli F, et al.

·  Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain phosphatase by Phos-tag SDS-PAGE[J]. The FASEB Journal, 2016, 30(1 Supplement): 1209.1-1209.1，Walsh M P, MacDonald J A, Sutherland C.

·  Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation[J]. Kidney Research: Experimental Protocols, 2016: 267-277，Horinouchi T, Terada K, Higashi T, et al.

·  The Abundance of Nonphosphorylated Tau in Mouse and Human Tauopathy Brains Revealed by the Use of Phos-Tag Method[J]. The American journal of pathology, 2016, 186(2): 398-409，Kimura T, Hatsuta H, Masuda-Suzukake M, et al.

·  Phos-tag SDS-PAGE resolves agonist-and isoform-specific activation patterns for PKD2 and PKD3 in cardiomyocytes and cardiac fibroblasts[J]. Journal of Molecular and Cellular Cardiology, 2016，Qiu W, Steinberg S F.

·  Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE[J]. American Journal of Physiology-Cell Physiology, 2016, 310(8): C681-C691，Sutherland C, MacDonald J A, Walsh M P.

·  Electrochemical biosensor for protein kinase A activity assay based on gold nanoparticles-carbon nanospheres, phos-tag-biotin and β-galactosidase[J]. Biosensors and Bioelectronics, 2016, 86: 508-515，Zhou Y, Yin H, Li X, et al.

·  Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE[J]. PloS one, 2016, 11(2): e0148294，Kinoshita-Kikuta E, Kinoshita E, Eguchi Y, et al.

·  Phosphopeptide Detection with Biotin-Labeled Phos-tag[J]. Phospho-Proteomics: Methods and Protocols, 2016: 17-29，Kinoshita-Kikuta E, Kinoshita E, Koike T.

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·  Microtubules Inhibit E-Cadherin Adhesive Activity by Maintaining Phosphorylated p120-Catenin in a Colon Carcinoma Cell Model[J]. PloS one, 2016, 11(2): e0148574，Maiden S L, Petrova Y I, Gumbiner B M.

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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