利用磁珠富集磷酸化蛋白

Phos-tag™ MG-Bead

Phos-tag™ MG-Bead是 phos-tag与涂有琼脂糖的磁珠连接而成的产品。磁珠上的phos-tag与磷酸化蛋白结合后，通过使用磁力架吸附磁珠即可完成富集。除蛋白外，还可以富集其他带有磷酸基团的物质。

使用此产品的论文

用于磷酸化SARS-CoV-2 核衣壳蛋白的分离

Yoko, I. et al. : Journal of Proteomics., 255 104501 (2022)

◆使用方法

1. 将5 ~ 50 µL Phos-tagTM MG-Bead磁珠加入1.5 mL试管中。

2. 使用磁力架收集管内磁珠，并去除保存液。

3. 用0.1 M Bis-tris-AcOH缓冲液使磁珠重悬，并摇匀。重复此步骤两次。

4. 取50 ~ 200 µL溶解于0.1 M Bis-tris-AcOH缓冲液的样品于1.5 mL试管中，并在室温下摇匀孵育 3 分钟。

5. 用磁力架收集管内磁珠，并去除穿透液。

6. 使用0.1 M Bis-tris-AcOH缓冲液重悬磁珠，摇匀30秒，用磁力架收集管内磁珠，并去除洗涤液。重复此步骤三次。

7. 用蒸馏水使磁珠重悬，摇匀30秒。震荡后，用磁力架收集管内磁珠，去除洗涤液。

8. 为洗脱与磁珠结合的磷酸化化合物，加入pyrophoshate缓冲液，并摇匀30 秒。重复此步骤五次。

9. 分析洗脱的磷酸化化合物。

*关于操作所需的相关溶液成分，请点击参阅本产品的protocol。

◆应用实例

富集磷酸化肽段

磁珠量：50 µL

样品：胰蛋白酶消化的 β-酪蛋白溶液（包括具有 1 个磷酸化位点和 4 个磷酸化位点的肽）

洗涤缓冲液：0.1 M Bis-tris-AcOH （含0.1 M NaCl） pH6.8

洗脱缓冲液：0.1 M Na₄P₂O₇ – 0.1M AcOH pH7.0

分别对样品溶液（sample）、穿透液（FT），洗脱液（E1）进行HPLC分析，结果如图所示，在洗脱液（E1）中成功检出两种磷酸化肽、。

FT：上述操作步骤5中的.穿透液。

E1：上述操作步骤8的洗脱液。

分离NAD和NADP

磁珠量：50 µL

样品：含有 NAD(100 nmol) 和 NADP(100 nmol) 的溶液

洗涤缓冲液：0.1 M Bis-tris-AcOH pH6.8

洗脱缓冲液：0.1 M Na₄P₂O₇ – 0.1M AcOH pH7.0

分别对各步骤的溶液进行HPLC分析，并对其结果进行解析。如图，穿透液和洗涤液中检测出NAD，通过反复洗涤，可以分离出高纯度的NAD。并在洗脱液检测出NADP，通过重复洗脱步骤，可以分离出纯度超过 99% 的NADP。

FT：上述操作步骤5的穿透液。

W1~W3：上述操作步骤6.的洗涤液。数字为洗涤次数。

W4：上述操作步骤7.的洗涤液。

◆相关产品

磁珠捕获磁力架

Phos-tag™ 丙烯酰胺

SuperSep Phos-tag™ 预制胶

点击此处查看产品系列

点击此处查看产品宣传页

※ 本页面产品仅供研究用，研究以外不可使用。

Phos-tag™ MG-Bead Protocol

Phos-tag™ 凝胶荧光染料

Phos-tag™ 凝胶荧光染料，是一种可在生理pH范围内，对凝胶中的磷酸化蛋白进行染色的荧光染料。进行了SDS-PAGE，用本产品处理聚丙烯酰胺凝胶，可以对磷酸化蛋白进行特异性染色。

本系列有波长不同的 Yellow（黄）、Magenta（品红）、Cyan（蓝绿）、Aqua（浅绿）四种颜色。每管规格为 0.2 mg，可对约20个迷你凝胶进行染色。

购买前请注意：

使用Phos-tag^™ 凝胶荧光染料，需要用到 Mixed reagents for Phos-tag™ Common Solution 5×（产品编号：383-15231）。请一同购买。

◆特点

●　高灵敏度

●　在生理pH下即可染色

●　选择性与pSer、 pTyr、 pHis以及pAsp残基结合

●　无需放射性物质、化学物质标签、抗体

●　2小时内完成染色

◆产品对比

◆应用实例①

磷酸化蛋白与非磷酸化蛋白的染色 Phos-tag™ Aqua（浅绿）

Lane.No.（）内为磷酸基团数目

Phos-tag™ Aqua（浅绿）染料选择性：用Phos-tag™ Aqua（浅绿）对10％（w/v）的PAGE胶进行染色，其中分别含有四组完整的以及经ALP（碱性磷酸酶）处理的磷酸蛋白（α-酪蛋白，β-酪蛋白，卵清蛋白和胃蛋白酶各1 μg）（左），然后使用考马斯亮蓝CBB G-250染色（右）。

成功特异性地检测出SDS-PAGE后的磷酸化蛋白。

数据提供：广岛大学研究生院 医学系科学研究科 医药分子机能科学研究室 木下惠美子老师、木下英司老师、小池透老师

◆应用实例②

使用Phos-tag™ 荧光凝胶染料的组氨酸、天门冬氨酸的磷酸化分析

细菌通过将信息从组氨酸激酶EnvZ传递到应答调控子OmpR来调节基因的表达。分别分析有自磷酸化能力的EnvZ激酶和被EnvZ催化的OmpR中组氨酸和天门冬氨酸的磷酸化。

可以确认Phos-tag™ Magenta（品红）和Phos-tag™ SDS-PAGE 这两种试剂中的His和Asp的磷酸化随激酶的处理时间经过不断增强。尤其是使用Phos-tag™ Magenta（品红）时，可以仅检测出His磷酸化或Asp磷酸化。

数据提供：广岛大学研究生院 医学系科学研究科 医药分子机能科学研究室 木下惠美子老师、木下英司老师、小池透老师

◆应用实例③

用于筛选组氨酸激酶抑制剂（新型抗生素）

使用Phos-tag™荧光凝胶染料，筛选组氨酸激酶抑制剂。

证实磷酸化依赖组氨酸激酶抑制剂浓度被抑制。

数据提供：广岛大学研究生院 医学系科学研究科 医药分子机能科学研究室 木下惠美子老师、木下英司老师、小池透老师

参考文献

“Quantitativemonitoring of His and Asp phosphorylation in a bacterial signaling system byusing Phos-tag Magenta/Cyan fluorescent dyes”, Emiko Kinoshita-Kikuta, HiroshiKusamoto, Syogo Ono, Keisuke Akayama, Yoko Eguchi, Masayuki Igarashi, ToshihideOkajima, Ryutaro Utsumi, Eiji Kinoshita, Tohru Koike, Electrophoresis, 2019

◆使用方法

※调整平衡及清洗溶液以及染色溶液必须使用Mixed reagents for Phos-tag™ Common Solution 5× (产品编号：383-15231)。

◆激发波长/荧光波长

Phos-tag™ Yellow（黄）	Phos-tag™ Magenta（品红）
Ex/Em=505 nm/514 nm	Ex/Em=547 nm/561 nm
Phos-tag™ Cyan（蓝绿）	Phos-tag™ Aqua（浅绿）
Ex/Em=643 nm/661nm	Ex/Em=551 nm/564 nm

◆产品列表

◆相关产品

Phos-tag™ Acrylamide

SuperSep™ Phos-tag™

用于Bio-Rad伯乐电泳仪

用于Life Technologies伯乐电泳仪

Phos-tag™ Biotin

Phos-tag™ Agarose

Phos-tag™ Tip

Phos-tag™ Mass Analytical Kit

※ 本页面产品仅供研究用，研究以外不可使用。

Phos-tag™ 琼脂糖枪头

Phos-tag™ Tip

　　可特异性捕捉磷酸基团的功能性分子“Phos-tag ™”的磷酸化肽纯化用枪头。

　　枪头里含有 Phos-tag ™ 琼脂糖，是即开即用的前处理工具，适用于生理状态下低分子量磷酸化分子（核酸和多肽等）的分离和富集。

◆原理

把 Phos-tag™ Tip 装在注射器（购买产品附送）上使用。

Phos-tag ™ Tip 的结构

◆优点、特色

◆案例、应用

分离磷酸化肽使用例

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

◆原理：

◆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

◆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：

Phos-tag 第6版说明书

Phos-tag系列 ver. 8

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·  Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability[J]. Molecular Cell, 2016，Spies J, Waizenegger A, Barton O, et al.

·  PhostagTM-gel retardation and in situ thylakoid kinase assay for determination of chloroplast protein phosphorylation targets[J]. Endocytobiosis and Cell Research, 2016, 27(2): 62-70，Dytyuk Y, Flügge F, Czarnecki O, et al.

·  Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis[J]. Biology of reproduction, 2016: biolreprod. 115.135897，Egbert J R, Uliasz T F, Shuhaibar L C, et al.

·  Newby, AC, & Bond, M.(2016). The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP[J]. Journal of Molecular and Cellular Cardiology, 2016, 90: 1-10，Kimura-Wozniak T, Duggirala A, Smith M C, et al. G.

·  Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels[J]. The FEBS Journal, 2016, 283(15): 2911-2928，Toume M, Tani M.

·  Regulation of CsrB/C sRNA decay by EIIAGlc of the phosphoenolpyruvate: carbohydrate phosphotransferase system[J]. Molecular microbiology, 2016, 99(4): 627-639，Leng Y, Vakulskas C A, Zere T R, et al.

·  The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint[J]. Molecular and cellular biology, 2016: MCB. 00952-15，Negishi T, Veis J, Hollenstein D, et al.

·  Validation of chemical compound library screening for transcriptional co‐activator with PDZ‐binding motif inhibitors using GFP‐fused transcriptional co‐activator with PDZ‐binding motif[J]. Cancer science, 2016, 107(6): 791-802，Nagashima S, Maruyama J, Kawano S, et al.

·  ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy[J]. Molecular cell, 2016, 62(3): 359-370，Li T Y, Sun Y, Liang Y, et al.

·  Spatiotemporal dynamics of Oct4 protein localization during preimplantation development in mice[J]. Reproduction, 2016: REP-16-0277，Fukuda A, Mitani A, Miyashita T, et al.

·  The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress[J]. Parasites & Vectors, 2016, 9(1): 1，Pastor-Fernández I, Regidor-Cerrillo J, Álvarez-García G, et al.

·  Interaction Analysis of a Two-Component System Using Nanodiscs[J]. PloS one, 2016, 11(2): e0149187，Hörnschemeyer P, Liss V, Heermann R, et al.

·  Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy[J]. Journal of Biological Chemistry, 2016, 291(31): 16162-16174，Akabane S, Matsuzaki K, Yamashita S, et al.

·  p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261[J]. Molecular and cellular biology, 2016, 36(10): 1540-1554，Beenstock J, Melamed D, Mooshayef N, et al.

·  Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids[J]. The FASEB Journal, 2016, 30(5): 2027-2039，Akagi S, Kono N, Ariyama H, et al.

·  Characterization of a herpes simplex virus 1 (HSV-1) chimera in which the Us3 protein kinase gene is replaced with the HSV-2 Us3 gene[J]. Journal of virology, 2016, 90(1): 457-473，Shindo K, Kato A, Koyanagi N, et al.

·  Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping[J]. FEBS letters, 2016, 590(10): 1530-1542，George S, Aguirre J D, Spratt D E, et al.

·  Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris[J]. PLoS Genet, 2016, 12(3): e1005922，Ma P, Mori T, Zhao C, et al.

·  Phosphorylation of Bni4 by MAP kinases contributes to septum assembly during yeast cytokinesis[J]. FEMS Yeast Research, 2016, 16(6): fow060，Pérez J, Arcones I, Gómez A, et al.

·  Alteration of Antiviral Signalling by Single Nucleotide Polymorphisms (SNPs) of Mitochondrial Antiviral Signalling Protein (MAVS)[J]. PloS one, 2016, 11(3): e0151173，Xing F, Matsumiya T, Hayakari R, et al.

·  Arm-in-arm response regulator dimers promote intermolecular signal transduction[J]. Journal of bacteriology, 2016, 198(8): 1218-1229，Baker A W, Satyshur K A, Morales N M, et al.

·  The lsh/ddm1 homolog mus-30 is required for genome stability, but not for dna methylation in neurospora crassa[J]. PLoS Genet, 2016, 12(1): e1005790，Basenko E Y, Kamei M, Ji L, et al.

·  Fine tuning chloroplast movements through physical interactions between phototropins[J]. Journal of Experimental Botany, 2016: erw265，Sztatelman O, Łabuz J, Hermanowicz P, et al.

·  Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle[J]. Parasitology, 2016, 143(01): 97-113，Pastor-Fernandez I, Regidor-Cerrillo J, Jimenez-Ruiz E, et al.

·  Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness[J]. Applied and environmental microbiology, 2016, 82(10): 2929-2942，Yasugi M, Okuzaki D, Kuwana R, et al.

·  Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae[J]. Genetics, 2016: genetics. 115.183939，Paulissen S M, Slubowski C J, Roesner J M, et al.

·  DDK dependent regulation of TOP2A at centromeres revealed by a chemical genetics approach[J]. Nucleic Acids Research, 2016: gkw626，Wu K Z L, Wang G N, Fitzgerald J, et al.

·  OVATE Family Protein 8 Positively Mediates Brassinosteroid Signaling through Interacting with the GSK3-like Kinase in Rice[J]. PLoS Genet, 2016, 12(6): e1006118，Yang C, Shen W, He Y, et al.

·  Epithelial Sel1L is required for the maintenance of intestinal homeostasis[J]. Molecular biology of the cell, 2016, 27(3): 483-490， Sun S, Lourie R, Cohen S B, et al.

·  Effect of Sodium Dodecyl Sulfate Concentration on Supramolecular Gel Electrophoresis[J]. ChemNanoMat, 2016，Tazawa S, Kobayashi K, Yamanaka M.

·  Intergenic VNTR Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes[J]. Infection and immunity, 2016: IAI. 00258-16，Zhu L, Olsen R J, Horstmann N, et al.

·  Ajuba Phosphorylation by CDK1 Promotes Cell Proliferation and Tumorigenesis[J]. Journal of Biological Chemistry, 2016: jbc. M116. 722751，Chen X, Stauffer S, Chen Y, et al.

·  Editorial: International Plant Proteomics Organization (INPPO) World Congress 2014[J]. Frontiers in Plant Science, 2016, 7，Heazlewood J L, Jorrín-Novo J V, Agrawal G K, et al.

·  Phosphoinositide kinase signaling controls ER-PM cross-talk[J]. Molecular biology of the cell, 2016, 27(7): 1170-1180，Omnus D J, Manford A G, Bader J M, et al.

·  A multiple covalent crosslinked soft hydrogel for bioseparation[J]. Chemical Communications, 2016, 52(15): 3247-3250，Liu Z, Fan L, Xiao H, et al.

·  Advances in crop proteomics: PTMs of proteins under abiotic stress[J]. Proteomics, 2016, 16(5): 847-865，Wu X, Gong F, Cao D, et al.

·  Cyclin-Dependent Kinase Co-Ordinates Carbohydrate Metabolism and Cell Cycle in S. cerevisiae[J]. Molecular cell, 2016, 62(4): 546-557，Zhao G, Chen Y, Carey L, et al.

·  Carbon Monoxide Gas Is Not Inert, but Global, in Its Consequences for Bacterial Gene Expression, Iron Acquisition, and Antibiotic Resistance[J]. Antioxidants & redox signaling, 2016，Wareham L K, Begg R, Jesse H E, et al.

·  Two-layer regulation of PAQR3 on ATG14-linked class III PtdIns3K activation upon glucose starvation[J]. Autophagy, 2016: 1-2，Xu D, Wang Z, Chen Y.

·  Regulation of sphingolipid biosynthesis by the morphogenesis checkpoint kinase Swe1[J]. Journal of Biological Chemistry, 2016, 291(5): 2524-2534，Chauhan N, Han G, Somashekarappa N, et al.

·  PAX5 tyrosine phosphorylation by SYK co-operatively functions with its serine phosphorylation to cancel the PAX5-dependent repression of BLIMP1: A mechanism for antigen-triggered plasma cell differentiation[J]. Biochemical and biophysical research communications, 2016, 475(2): 176-181，Inagaki Y, Hayakawa F, Hirano D, et al.

·  A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock[J]. Journal of Bacteriology, 2016: JB. 00235-16，Boyd J S, Cheng R R, Paddock M L, et al.

·  HuR mediates motility of human bone marrow-derived mesenchymal stem cells triggered by sphingosine 1-phosphate in liver fibrosis[J]. Journal of Molecular Medicine, 2016: 1-14，Chang N, Ge J, Xiu L, et al.

·  Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts[J]. Biochemistry and Biophysics Reports, 2016，Kitakaze K, Tasaki C, Tajima Y, et al.

·  Roseotoxin B Improves Allergic Contact Dermatitis through a Unique Anti-inflammatory Mechanism Involving Excessive Activation of Autophagy in Activated T-Lymphocytes[J]. Journal of Investigative Dermatology, 2016，Wang X, Hu C, Wu X, et al.

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

  
  

Phos-tag™ Agarose

亲和层析纯化磷酸化蛋白

◆原理

◆优点、特色

●　无需使用还原剂或表面活性剂即可纯化

●　与亲和层析方法类似。

●　可在１小时内纯化磷酸化蛋白。

●　Phos-tag™ Agarose 捕获结合到 Tyr、Thr、Ser、Asp、His 等氨基酸、糖类、脂类上的无机磷酸根和大量二价磷酸根。

●　可在生理条件下（pH7.5）捕捉蛋白。

●　纯化后的产物可用于 Co-IP 实验和其他蛋白活性实验。

◆案例、应用：

【使用例子：A431 裂解液中的磷酸化蛋白的纯化】

把Phos-tag™ 填充到柱里，再加上 A431 裂解液。

SYPRO Ruby 染色（左图）再使用 Anti-p Tyr 抗体进行免疫印迹（右图），检测出结果。

结果确认磷酸化蛋白浓缩在柱吸附层里。

M：分子量标记

Lane 1：未吸附层

Lane 2：吸附层

Lane 3：柱清洗层

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

◆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6版说明书

Phos-tag系列 ver. 8

说明书

【参考文献】

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·  PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7[J]. Nature communications, 2016, 7，Shinde S R, Maddika S.

·  Feedback control of ErbB2 via ERK-mediated phosphorylation of a conserved threonine in the juxtamembrane domain[J]. Scientific Reports, 2016, 6: 31502，Kawasaki Y, Sakimura A, Park C M, et al.

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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.

·  A Phos‐tag SDS‐PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1[J]. Proteomics, 2016，Kinoshita E, Kinoshita‐Kikuta E, Kubota Y, et al.

·  Difference gel electrophoresis of phosphoproteome: U.S. Patent Application 15/004,339[P]. 2016-1-22，Tao W A, Wang L.

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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.

·  Serine 231 and 257 of Agamous-like 15 are phosphorylated in floral receptacles[J]. Plant Signaling & Behavior, 2016, 11(7): e1199314，Patharkar O R, Macken T A, Walker J C.

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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

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