◆案例、應用

【使用Phos-tag™ SDS-PAGE的磷酸化/非磷酸化蛋白比較】

我推薦使用Phos-tag ™ ——東京大學研究院醫(yī)學研究科 小川覺之

　　Phos-tag ™ 是專為研究磷酸化蛋白而新開發(fā)出來的試劑。此產(chǎn)品使用方便，不但可用于體外實驗，還能定量分析體內(nèi)蛋白的磷酸化水平。Phos-tag ™ SDS-PAGE 可用于常規(guī)電泳實驗，無需購買特殊設(shè)備，性價比高。傳統(tǒng)蛋白磷酸化的研究需要特異的磷酸化抗體、RI 等其它試劑，操作復雜，花費大，且放射性元素會有安全隱患，而Phos-tag ™ 的出現(xiàn)恰恰可以彌補這些缺點，為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 的分離效果圖

　　Lane 1 為非磷酸化蛋白，Lane 2-5 為磷酸化蛋白，各蛋白因磷酸化狀態(tài)不同而條帶遷移率也有所不同。

　　磷酸化/ 非磷酸化蛋白的數(shù)量比、磷酸化程度、磷酸化蛋白的豐度等都可根據(jù)條帶遷移和條帶濃度求得。

【資料提供】

日本東京大學研究生院醫(yī)學系研究科

【二維電泳中的應用：分析hnRNP K 磷酸化異構(gòu)體】

　　小鼠巨噬細胞J774.1 經(jīng)LPS 刺激后，裂解細胞，經(jīng)過免疫沉淀法分離得到hnRNP K。在二維電泳中，一維是IPG 膠，二維是Phos-tag ™ SDS-PAGE，可分離hnRNP K 的異構(gòu)體。利用質(zhì)譜儀，可以確認不同的點代表不同的亞型或修飾蛋白。

二維電泳

　　同一個等電點的位置上，不同位點發(fā)生磷酸化都可以被區(qū)分開來（例: spots 6 vs. 8 and spots 4 vs. 7）

【參考文獻】

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.

【結(jié)果提供】

　　橫濱市立大學 生命納米系統(tǒng)科學研究科 生物體超分子系統(tǒng)科學專業(yè) 木村彌生（Dr. Y. Kimura）、平野久（Dr. H. Hirano）理化學研究所RCAI 小原收

【EGF 刺激前后MAPK 磷酸化水平的變化】

　　常規(guī)SDS-PAGE 和Phos-tagTM SDS-PAGE 后迚行克疫印跡實驗分析EGF 刺激的A431 細胞中MAPK 磷酸化水平。

　　摘自Kinoshita-Kikuta, E. et al., Mol.Cell. Proteomics. (2007)6: 356.

產(chǎn)品編號	產(chǎn)品名稱	產(chǎn)品規(guī)格	產(chǎn)品等級	產(chǎn)品價格
304-93526	?Phos-tag?Acrylamide?AAL-107 5mM?Aqueous?Solution?Phos-tag?丙烯酰胺5mM水溶液	0.3mL	蛋白研究	–
300-93523	?Phos-tag?Acrylamide?AAL-107 Phos-tag?丙烯酰胺	2mg	蛋白研究	–
304-93521	?Phos-tag?Acrylamide?AAL-107 Phos-tag?丙烯酰胺	10mg	蛋白研究	–
134-15302	Manganese(II)?Chloride?Tetrahydrate氯化錳四水合物	25g	for?Molecular?Biology	–

Phos-tag™ 系列磷酸化蛋白新方法！

　　Phos-tag™是一種能與磷酸離子特異性結(jié)合的功能性分子。它可用于磷酸化蛋白的分離（Phos-tag™ Acrylamide）、Western Blot檢測（Phos-tag™ Biotin）、蛋白純化 （Phos-tag™Agarose）及質(zhì)譜分析MALDI-TOF/MS （Phos-tag™ Mass Analytical Kit）。

◆Phos-tag™ 的基本結(jié)構(gòu)

特點：

與-2價磷酸根離子的親和性和選擇性高于其它陰離子

在pH 5-8的生理環(huán)境下生成穩(wěn)定的復合物

◆原理

 

◆相關(guān)應用

◆相關(guān)產(chǎn)品

產(chǎn)品名稱	用　　途
Phos-tag™ Acrylamide	分離: SDS – PAGE 分離不同磷酸化水平的蛋白
SuperSep Phos-tag™	分離: 預制膠中含有50μM Phos-tag™ Acrylamide
Phos-tag™ Biotin	檢測: 代替 Western Blot 檢測中的磷酸化抗體
Phos-tag™ Agarose	純化: 通用柱層析，純化磷酸化蛋白
Phos-tag™ Mass Analytical Kit	分析: 用于質(zhì)譜 MALDI-TOF/MS 分析，提高磷酸化分子的檢測靈敏度

phos-tag™由日本廣島大學研究生院醫(yī)齒藥學綜合研究科醫(yī)藥分子功能科學研究室開發(fā)。

更多產(chǎn)品信息，請點擊：http://phos-tag.jp

1.???? Phos-tag? Acrylamide的溶解

5mmmol/ Phos-tag? 液體 (3v/v% 甲醇）：

1)??? 10mg? Phos-tag? Acrylamide 里加入 0.1mL 甲醇

2)??? 使用槍頭攪拌混合直至完全溶解。

3)??? ?加3.2mL 蒸餾水， 用槍頭混勻。

2-8℃避光保存。不適合零度以下保存。建議保存時間6個月。

　　?注意：避免溶解過程出現(xiàn)白色懸浮顆粒。

2.???? α-Casein, from Bovine Milk, Dephosphorylated（038-23221），陽性對照（含有磷酸化和非磷酸化

　　?α-Casein），如何使用？

　　?用水或者上樣buffer溶解。用水溶解后，冷凍保存。電泳條件：Phos-tag? 50umol/L,分離膠濃度 10%。

　　?電流：30mM，1小時。

3.???? 用Alkaline Phosphatase(for Biochemistry)（018-10693）進行磷酸化蛋白的去磷酸化反應體系。

37℃，過夜。# 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有一點需要注意:ALP活性化使用Mg離子，相

　　?同的非磷酸化蛋白質(zhì)用ALP處理的樣品的條帶和沒有用ALP處理的樣品的條帶的位置不同。

4.???? Phos-tag? SDS-PAGE實驗沒有成功分離磷酸化蛋白：

1） 使用α-Casein, from Bovine Milk, Dephosphorylated（038-23221）作為陽性對照，確認實驗條

件和試劑均沒有問題。

2） 可使用Phos-tag?Biotin檢測樣品中是否有磷酸化蛋白。確認有磷酸化蛋白后，再通過

Phos-tag ?SDS-PAGE進行分離鑒定。

3） 經(jīng)質(zhì)譜鑒定有表達磷酸化蛋白，建議增大樣品的含量，可使用Phos-tag ?Agarose進行磷酸化蛋白

的富集。磷酸化蛋白含量過低，會影響其分離效果。

4） 文獻報道有表達磷酸化蛋白，或者同源蛋白有表達磷酸化蛋白的，建議用Phos-tag? Biotin先確認

樣品中是否有磷酸化蛋白。

5） 建議樣品的pH值在7左右。酸性或者堿性條件下，Mn2⁺-Phos-tag?與磷酸化基團的特異性結(jié)合較

差。

6） 避免樣品中含有高濃度的還原劑，變性劑，表面活性劑等。β-巰基乙醇濃度不高于0.2M（或者5%）。

7） 進行Phos-tag? SDS-PAGE的佳樣品是純化的蛋白。如果是細胞裂解液，體外激酶反應液，組織均

漿液等，需要摸索佳的分離膠，Phos-tag? Acylamide的濃度。建議Phos-tag? Acrylamide濃度

從50uM開始摸索。

5.???? Phos-tag?SDS-PAGE凝膠用于Western Blotting實驗的優(yōu)化建議：

1） 可以檢測的樣品包括體外激酶反應體系，細胞裂解液，組織均漿液。

2） 每孔樣品的上樣量是10~30ug（請根據(jù)蛋白表達量進行調(diào)整）

3） 制備樣品中含有的還原劑、變性劑、螯合劑、釩酸等會使電泳條帶發(fā)生彎曲或者拖尾。通過TCA沉淀或

滲析法降低雜質(zhì)含量。

4） 建議樣品的pH值在7左右。如果加入上樣緩沖液后溶液顯黃色或者橙色，加入Tris緩沖液調(diào)整pH值為7。

5） 目的蛋白分子量大于60kDa，分離膠的丙烯酰胺濃度為6%；目的蛋白分子量小于60kDa,分離膠的丙烯

酰胺濃度為8%。

6） 如果樣品中含有大量蛋白，比如細胞裂解液，組織均漿液，Phos-tag? Acylamide濃度為5~25uM。

若目的蛋白濃度低，建議Phos-tag? Acylamide濃度為100uM。

7） Phos-tag ?SDS-PAGE凝膠用于Western Blotting實驗，濕法轉(zhuǎn)膜建議：10mM EDTA的轉(zhuǎn)移緩沖液

處理凝膠10min，不含有EDTA的轉(zhuǎn)移緩沖液處理凝膠10min。重復3次。強烈建議濕法轉(zhuǎn)膜

8） Phos-tag? SDS PAGE半干法轉(zhuǎn)膜建議：

????????????? i.????? 電泳后用含有EDTA的轉(zhuǎn)移緩沖液處理凝膠，EDTA的濃度為 100mM。100mM EDTA的轉(zhuǎn)移

緩沖液處理凝膠10min，不含有EDTA的轉(zhuǎn)移緩沖液處理凝膠10min。重復3次。

???????????? ii.????? 轉(zhuǎn)膜的電流值提高2%~3%, 延長時間2成。

??????????? iii.????? 轉(zhuǎn)膜的緩沖液加SDS，加到大約0.05~0.2%，轉(zhuǎn)膜效率會提高。

9） 使用目的蛋白的非磷酸化抗體即可。比如檢測各種腫瘤細胞系中Src激酶活性實驗，用Src的非磷酸化抗

體即可。

10） 和光的WIDE-VIEW^TM Prestained Protein Siza MarkerIII(230-02461)可檢測作為轉(zhuǎn)膜效率，但是無

法判斷分子量。

11） 一般預染的蛋白marker在Phos-tag?SDS-PAGE中條帶會彎曲，無法判斷蛋白分子量。

12） 不能確認磷酸化蛋白和非磷酸化蛋白的分離，請進行常規(guī)的SDS-PAGE，Western Blotting實驗。比

對目的蛋白的遷移率。

13） 不能確認是因為蛋白發(fā)生磷酸化還是出現(xiàn)降解造成蛋白條帶遷移，請進行常規(guī)的SDS-PAGE實驗，確

認不會出現(xiàn)條帶遷移。

14） 目的蛋白磷酸化與非磷酸化分離效果不佳，使用α-Casein, from Bovine Milk, Dephosphorylated

（038-23221）作為陽性對照，確認實驗條件和試劑均沒有問題。如果確認能夠分離，調(diào)整分離膠，

Phos-tag? Acylamide的濃度。建議使用品質(zhì)佳的MnCl2（139-00722）。

【參考文獻】

· ?Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling[J].Nature communications, 2016, 7，Shindo Y, Iwamoto K, Mouri K, et al.

· ?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.

· ?Plastid-nucleus communication involves calcium-modulated MAPK signalling[J]. Nature Communications, 2016, 7，Guo H, Feng P, Chi W, et al.

· ?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.

· ?ERK1/2-induced phosphorylation of R-Ras GTPases stimulates their oncogenic potential[J]. Oncogene, 2016，F(xiàn)rémin C, Guégan J P, Plutoni C, et al.

· ?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.

· ?A small molecule pyrazolo [3, 4-d] pyrimidinone inhibitor of zipper-interacting protein kinase suppresses calcium sensitization of vascular smooth muscle[J]. Molecular pharmacology, 2016, 89(1): 105-117，MacDonald J A, Sutherland C, Carlson D A, et al.

· ?The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2/CPL1 interacts with eIF4AIII and is essential for nonsense-mediated mRNA decay in Arabidopsis[J]. The Plant Cell, 2016: TPC2015-00771-RA，Chen T, Qin T, Ding F, et al.

· ?Vasorelaxant Effect of 5′-Methylthioadenosine Obtained from Candida utilis Yeast Extract through the Suppression of Intracellular Ca2+ Concentration in Isolated Rat Aorta[J]. Journal of agricultural and food chemistry, 2016, 64(17): 3362-3370，Kumrungsee T, Akiyama S, Saiki T, et al.

· ?Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells[J]. Biochemical and biophysical research communications, 2016, 472(4): 598-602，Nakashima A, Ohnuma S, Kodani Y, et al.

· ?Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity[J]. The Journal of Neuroscience, 2016, 36(19): 5299-5313，Bertling E, Englund J, Minkeviciene R, et al.

· ?AMPK-dependent phosphorylation of lipid droplet protein PLIN2 triggers its degradation by CMA[J]. Autophagy, 2016, 12(2): 432-438，Kaushik S, Cuervo A M.

· ?Myocardin-related transcription factor a and yes-associated protein exert dual control in G protein-coupled receptor-and RhoA-mediated transcriptional regulation and cell proliferation[J]. Molecular and cellular biology, 2016, 36(1): 39-49，Olivia M Y, Miyamoto S, Brown J H.

· ?Extensive phosphorylation of AMPA receptors in neurons[J]. Proceedings of the National Academy of Sciences, 2016, 113(33): E4920-E4927，Diering G H, Heo S, Hussain N K, et al.

· ?The transmembrane region of guard cell SLAC1 channels perceives CO2 signals via an ABA-independent pathway in Arabidopsis[J]. The Plant Cell, 2016, 28(2): 557-567，Yamamoto Y, Negi J, Wang C, et al.

· ?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 T E, Duggirala A, Smith M C, et al.

· ?Atg13 is essential for autophagy and cardiac development in mice[J]. Molecular and cellular biology, 2016, 36(4): 585-595，Kaizuka T, Mizushima N.

· ?The ChrSA and HrrSA two-component systems are required for transcriptional regulation of the hemA promoter in Corynebacterium diphtheriae[J]. Journal of Bacteriology, 2016: JB. 00339-16，Burgos J M, Schmitt M P.

· ?Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes[J]. Infection and Immunity, 2016, 84(7): 2086-2093，Zhu L, Olsen R J, Horstmann N, et al.

· ?Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy[J]. Reproductive Toxicology, 2016, 62: 62-70，Ejdesj? A, Brings S, Fleming T, et al.

· ?Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression[J]. Proceedings of the National Academy of Sciences, 2016, 113(23): 6490-6495，Chuang L S H, Khor J M, Lai S K, et al.

· ?Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence[J]. Journal of experimental botany, 2016: erw107，Cho H Y, Wen T N, Wang Y T, et al.

· ?Temporal regulation of lipin activity diverged to account for differences in mitotic programs[J]. Current Biology, 2016, 26(2): 237-243，Makarova M, Gu Y, Chen J S, et al.

· ?Block of CDK1‐dependent polyadenosine elongation of Cyclin B mRNA in metaphase‐i‐arrested starfish oocytes is released by intracellular pH elevation upon spawning[J]. Molecular reproduction and development, 2016, 83(1): 79-87，Ochi H, Aoto S, Tachibana K, et al.

· ?Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1[J]. Cell reports, 2016, 15(9): 2050-2062，Rodriguez-Rodriguez J A, Moyano Y, Játiva S, et al.

· ?PLK2 phosphorylates and inhibits enriched TAp73 in human osteosarcoma cells[J]. Cancer medicine, 2016, 5(1): 74-87，Hu Z B, Liao X H, Xu Z Y, et al.

· ?Phosphorylated TDP-43 becomes resistant to cleavage by calpain: A regulatory role for phosphorylation in TDP-43 pathology of ALS/FTLD[J]. Neuroscience research, 2016, 107: 63-69，Yamashita T, Teramoto S, Kwak S.

· ?The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects[J]. Nucleic acids research, 2016: gkw506，Herruzo E, Ontoso D, González-Arranz S, et al.

· ?An optimized guanidination method for large‐scale proteomic studies[J]. Proteomics, 2016，Ye J, Zhang Y, Huang L, et al.

· ?Expression and purification of the kinase domain of PINK1 in Pichia pastoris[J]. Protein Expression and Purification, 2016，Wu D, Qu L, Fu Y, et al.

· ?BRI2 and BRI3 are functionally distinct phosphoproteins[J]. Cellular signalling, 2016, 28(1): 130-144，Martins F, Rebelo S, Santos M, et al.

· ?Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics[J]. Proteomics, 2016，Yang W, Jackson B, Zhang H.

· ?Regulation of Beclin 1 Protein Phosphorylation and Autophagy by Protein Phosphatase 2A (PP2A) and Death-associated Protein Kinase 3 (DAPK3)[J]. Journal of Biological Chemistry, 2016, 291(20): 10858-10866，F(xiàn)ujiwara N, Usui T, Ohama T, et al.

· ?Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL[J]. Biochemistry, 2016, 55(29): 4027-4035，Yamawaki T, Ishikawa H, Mizuno M, et al.

· ?Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2016, 1863(4): 650-659，Yang D, Okamura H, Teramachi J, et al.

· ?Analysis of Molecular Species Profiles of Ceramide-1-phosphate and Sphingomyelin Using MALDI-TOF Mass Spectrometry[J]. Lipids, 2016, 51(2): 263-270，Yamashita R, Tabata Y, Iga E, et al.

· ?Highly sensitive myosin phosphorylation analysis in the renal afferent arteriole[J]. Journal of Smooth Muscle Research, 2016, 52(0): 45-55，Takeya K.

· ?Functional dissection of the CroRS two-component system required for resistance to cell wall stressors in Enterococcus faecalis[J]. Journal of bacteriology, 2016, 198(8): 1326-1336，Kellogg S L, Kristich C J.

· ?Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle[J]. American Journal of Physiology-Cell Physiology, 2016, 310(11): C921-C930，Trappanese D M, Sivilich S, Ets H K, et al.

· ?ModProt: a database for integrating laboratory and literature data about protein post-translational modifications[J]. Journal of Electrophoresis, 2016, 60(1): 1-4，Kimura Y, Toda T, Hirano H.

· ?The C-ETS2-TFEB Axis Promotes Neuron Survival under Oxidative Stress by Regulating Lysosome Activity[J]. Oxidative medicine and cellular longevity, 2016,Ma S, Fang Z, Luo W, et al.

· ?Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions[J]. Photosynthesis Research, 2016: 1-10，Marutani Y, Yamauchi Y, Higashiyama M, et al.

· ?Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2016, 1863(1): 10-18，Jeon Y H, Ko K Y, Lee J H, et al.

· ?Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK[J]. BioMetals, 2016, 29(4): 715-729，F(xiàn)ojtikova V, Bartosova M, Man P, et al.

· ?Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein[J]. Archives of virology, 2016: 1-14，Qiu X, Zhan Y, Meng C, et al.

· ?Increased level of phosphorylated desmin and its degradation products in heart failure[J]. Biochemistry and Biophysics Reports, 2016, 6: 54-62，Bouvet M, Dubois-Deruy E, Alayi T D, et al.

· ?Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks[J]. Proceedings of the National Academy of Sciences, 2016: 201602827，Zhou C, Elia A E H, Naylor M L, et al.

· ?Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[J]. Nucleic Acids Research, 2016: gkw642，Brosse A, Korobeinikova A, Gottesman S, et al.

· ?Evolution of ZnII–Macrocyclic Polyamines to Biological Probes and Supramolecular Assembly[J]. Macrocyclic and Supramolecular Chemistry: How Izatt-Christensen Award Winners Shaped the Field, 2016: 415，Kimura E, Koike T, Aoki S.

· ?Phosphopeptide Enrichment Using Various Magnetic Nanocomposites: An Overview[J]. Phospho-Proteomics: Methods and Protocols, 2016: 193-209，Batalha í L, Roque A C A.

· ?In vivo phosphorylation of a peptide tag for protein purification[J]. Biotechnology letters, 2016, 38(5): 767-772，Goux M, Fateh A, Defontaine A, et al.

· ?Regulation of cell reversal frequency in Myxococcus xanthus requires the balanced activity of CheY‐like domains in FrzE and FrzZ[J]. Molecular microbiology, 2016，Kaimer C, Zusman D R.

· ?Elevation of cortical serotonin transporter activity upon peripheral immune challenge is regulated independently of p38 mitogen‐activated protein kinase activation and transporter phosphorylation[J]. Journal of neurochemistry, 2016, 137(3): 423-435，Schwamborn R, Brown E, Haase J.

· ?The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression[J]. Molecular cell, 2016, 62(4): 532-545，Ewald J C, Kuehne A, Zamboni N, et al.

· ?Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination[J]. Journal of Biological Chemistry, 2016, 291(9): 4649-4657，Takasugi T, Minegishi S, Asada A, et al.

· ?Increased level of phosphorylated desmin and its degradation products in heart failure[J]. Biochemistry and Biophysics Reports. 2016，Bouvet M, Dubois-Deruy E, Alayi T D, et al.

· ?a high‐affinity LCO‐binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor[J]. FEBS letters, 2016, 590(10): 1477-1487，F(xiàn)liegmann J, Jauneau A, Pichereaux C, et al. LYR3,

· ?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，F(xiàn)ukuda 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，H?rnschemeyer 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, ?abuz 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