蛋白磷酸化研究的預(yù)制膠

SuperSep Phos-tag™

　　SuperSep Phos-tag™ 是研究蛋白磷酸化的方法，無需特異性磷酸化抗體或者同位素標(biāo)記。

◆SuperSep Phos-tag™

　　Phos-tag™ 是一種預(yù)制膠，預(yù)先加入了50 μmol/L的Phostag™ Acrylamide，打開包裝即可直接使用。預(yù)制膠中含有鋅作為金屬離子，在中心凝膠緩沖液中保存穩(wěn)定性很好，得到的帶條結(jié)果也很整齊。

　　磷酸化蛋白和非磷酸化蛋白作為不同條帶分離。

　　分離后，膠可用于考馬斯亮藍(lán)染色，免疫印跡和質(zhì)譜實(shí)驗(yàn)。

已公開的驗(yàn)證蛋白列表，請點(diǎn)擊

◆Phos-tag™ SDS-PAGE 的原理

◆在HighWire Search 上搜索到的論文數(shù)

◆運(yùn)用

利用p35的丙氨酸突變體確定Cdk5 激活p35的磷酸化位點(diǎn)

　　p35常見的磷酸化位點(diǎn)是Ser8和Thr138。但是Ser8和Thr138位點(diǎn)往往會發(fā)生丙氨酸突變，產(chǎn)生3種突變體（Ser8突變體：S8A，Thr138突變體：T138A，Ser8和Thr138雙突變體：2A）。這3種突變體、野生型p35、Cdk5和沒有激酶活性的Cdk5都來源于COS-7細(xì)胞。這些細(xì)胞裂解液用Phos-tag™ SDS-PAGE和Western blotting 進(jìn)行檢測（檢測抗體：p35抗體）。

100 μM Phos-tag ™ 丙烯酰胺, 7.5% 聚丙烯酰胺凝膠

可明確磷酸化位點(diǎn)和條帶遷移率的關(guān)系！

– 泳道1（條帶L2和L4）和泳道5（條帶M1）：p35在Cdk5的作用下發(fā)生了磷酸化；

– 泳道1（條帶L2和L4）和泳道3（條帶L2和L4）：在無激酶活性Cdk5的作用下，大約有一半p35蛋白在Thr138位點(diǎn)

發(fā)生磷酸化，同樣在138位發(fā)生突變的p35蛋白亦是如此。

– 泳道5 （條帶M1）和泳道6（條帶L3和L4）：Ser8和Thr138是主要的磷酸化位點(diǎn)；

– 泳道5（條帶M1）、泳道7（條帶L1和L2）和泳道8（條帶M2）：條帶M1是Ser8和Thr138都發(fā)生磷酸化的條帶；

條帶M2是只有Ser8磷酸化的條帶；條帶L1和L2是只有Thr138磷酸化的條帶。

※ 條帶L1和L3中的X 是不確定哪個位點(diǎn)發(fā)生磷酸化的條帶；

※ 條帶L4是非磷酸化的p35。

【參考文獻(xiàn)】

▪ 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.

【結(jié)果提供】

理化學(xué)研究所 腦科學(xué)綜合研究中心 回路功能研究核心 記憶功能研究團(tuán)隊(duì) 細(xì)川智永（Dr. T. Hosokawa）

首都大學(xué)東京 理工學(xué)研究科 生命科學(xué)專業(yè) 神經(jīng)分子功能研究室 久永真市（Dr. S. Hisanaga）

檢測含有Dnmt1磷酸化激酶的片段

我們可以確定在片段中含有目的激酶！

① 采用親和色譜法從鼠腦提取液中純化GST-Dnmt1（1-290）結(jié)合蛋白

② 使用0.3 M 和1 M NaCl 的DNA 纖維素柱洗脫得到目的蛋白

③ GST-Dnmt1（1-290）作為體外激酶實(shí)驗(yàn)的反應(yīng)底物

④ Phos-tag ™ SDS-PAGE 用于Western blotting，確定遷移條帶中每個片段的激酶活性

【參考文獻(xiàn)】

▪ 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.,

【結(jié)果提供】

高知大學(xué) 綜合研究中心 生命、功能物質(zhì)部門 實(shí)驗(yàn)實(shí)習(xí)機(jī)器設(shè)施 杉山康憲（Dr. Y. Sugiyama）

香川大學(xué) 農(nóng)學(xué)部 應(yīng)用生物科學(xué)科 動物功能生化學(xué)研究室 龜下勇（Dr. I. Kameshita）

二維電泳中的應(yīng)用：分析hnRNP K磷酸化異構(gòu)體

　　小鼠巨噬細(xì)胞J774.1 經(jīng)LPS 刺激后，裂解細(xì)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(rèn)不同的點(diǎn)代表不同的亞型或修飾蛋白。

同一個等電點(diǎn)的位置上，不同位點(diǎn)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(xiàn)】

▪ 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(xué) 生命納米系統(tǒng)科學(xué)研究科 生物體超分子系統(tǒng)科學(xué)專業(yè) 木村彌生（Dr. Y. Kimura）、平野久（Dr. H. Hirano）

理化學(xué)研究所RCAI 小原收

◆備注

樣品制備：

Phos-tag SDS-PAGE對于蛋白樣品中的雜質(zhì)非常敏感，尤其是螯合劑，釩酸，無機(jī)鹽，表面活性劑這類物質(zhì)。

強(qiáng)烈建議在Phos-tag SDS-PAGE之前通過TCA沉淀或滲析法降低雜質(zhì)含量。

轉(zhuǎn)膜前處理：

另一個必須的步驟是在轉(zhuǎn)膜前，用EDTA去除凝膠中的金屬離子（Mn2+或者Zn2+）；

該步驟可提高蛋白的轉(zhuǎn)膜效率。

●　分別準(zhǔn)備10 mmol/L 含EDTA和不含EDTA 兩種1x transfer buffer。

●　將凝膠浸泡在含10 mmol/L EDTA的1x transfer buffer，至少20分鐘，溫和搖晃。更換新緩沖液，重復(fù)3次。

●　將凝膠浸泡在不含10 mmol/L EDTA的1x transfer buffer，10分鐘，溫和搖晃。

●　轉(zhuǎn)膜操作*。

* 建議用濕法轉(zhuǎn)膜，以提高轉(zhuǎn)膜效率。

◆質(zhì)量控制

　　每一批SuperSep Phos-tag™，出廠前均根據(jù)其產(chǎn)品規(guī)格進(jìn)行測試，以保證可分離磷酸化和非磷酸化蛋白，以及他們的分離成都在正常參數(shù)內(nèi)。

◆保存溫度

2-10℃

◆產(chǎn)品信息

用于Bio-Rad伯樂電泳儀

貨號	品名	電泳儀	規(guī)格
198-17981	SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 83×100×3.9mm	Mini-PROTEAN?  Tetra Cell (Bio-Rad Laboratories, Inc.)	5 塊
195-17991	SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 83×100×3.9mm		5 塊

用于Life Technologies電泳儀

貨號	品名	電泳儀	規(guī)格
192-18001	SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 100×100×6.6mm	XCell SureLock?  Mini-Cell (Life Technologies, Inc.)	5 塊
199-18011	SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 100×100×6.6mm		5 塊

用于Wako EasySeperator電泳儀

產(chǎn)品編號	產(chǎn)品	凝膠濃度	孔數(shù)	包裝
192-17401	SuperSep™ Phos-tag™ (50 μmol/L) Phos-tag™ 預(yù)制膠50 μmol/L	6.0%	13孔	5塊
199-17391		6.0%	17孔	5塊
195-17371		7.5%	13孔	5塊
192-17381		7.5%	17孔	5塊
193-16711		10.0%	13孔	5塊
190-16721		10.0%	17孔	5塊
195-16391		12.5%	13孔	5塊
193-16571		12.5%	17孔	5塊
193-16691		15.0%	13孔	5塊
196-16701		15.0%	17孔	5塊
197-16851		17.5%	13孔	5塊
194-16861		17.5%	17孔	5塊

◆相關(guān)產(chǎn)品

產(chǎn)品編號	產(chǎn)品名稱	規(guī)格
058-07681	EasySeparator Phos-tag預(yù)制凝膠的配套電泳槽	1 set

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(diǎn)：

與-2價(jià)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(fù)合物

◆原理：

◆相關(guān)應(yīng)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(yù)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(xué)研究生院醫(yī)齒藥學(xué)綜合研究科醫(yī)藥分子功能科學(xué)研究室開發(fā)。

更多產(chǎn)品信息，請點(diǎn)擊：http://phos-tag.jp

Phos-tag 第5版說明書

Phos-tag系列 ver 5

Q1.	我們可以采用哪種凝膠染色法？
A1.	所有的染色法都可使用，最常用的例如考馬斯亮藍(lán)染色法，負(fù)染，銀染和熒光染色等。
Q2.	用考馬斯亮藍(lán)染法染色，著色不明顯。
A2.	在微波爐內(nèi)進(jìn)行脫色，會取得比較滿意的效果。 方法：將染色的膠放在100毫升去離子水里，用擦拭紙包裹膠，再放進(jìn)微波爐加熱幾分鐘后更換去離子水，并重復(fù)上述步驟3或4次。請注意防止盛放膠的容器溫度過高。
Q3.	此款產(chǎn)品能否用于免疫印跡？
A3.	可以，如果用EDTA清除膠里面含有的鋅，可以提高轉(zhuǎn)膜的效率。 方法：膠浸在含有10 mmol/L EDTA的轉(zhuǎn)移緩沖液（25 mmol/L tris、192   mmol/L甘氨酸，10％甲醇）里輕輕攪拌10分鐘。重復(fù)上述步驟3次。然后放進(jìn)不含EDTA的轉(zhuǎn)移緩沖液（25 mmol/L tris、192   mmol/L的甘氨酸，10％甲醇）里攪拌10分鐘并轉(zhuǎn)移到PVDF膜或NC膜（硝酸纖維素膜）上。
Q4.	條帶扭曲了。
A4.	含有EDTA，無機(jī)鹽，表面活性劑等的樣品可能會導(dǎo)致條帶彎曲或者拖尾。通過TCA或透析沉淀脫鹽樣品。空白泳道也會導(dǎo)致相鄰樣品條帶彎曲，在空白泳道加與樣品相同體積的樣品緩沖液(x1)。
Q5.	磷酸化蛋白和非磷酸化蛋白不能分離。
A5.	將β–酪蛋白（038-23221）作為Phos-tag SDS-PAGE電泳的陽性對照，將用堿性磷酸酶處理的β–酪蛋白作為陰性對照，并檢查條帶的遷移。如果只有一個條帶，可能是由于Phos-tag™或丙烯酰胺的濃度沒有優(yōu)化導(dǎo)致磷酸化和非磷酸化蛋白不能分離。
Q6.	可用在細(xì)胞的粗提物上嗎？
A6.	可以的，可能Rf值可能有稍低，由于目的蛋白的因素，條帶可能會比較模糊。
Q7.	上樣量多少？
A7.	1至5微克純化的蛋白（考馬斯亮藍(lán)染法），10至30微克的組織或細(xì)胞提取物（取決于蛋白質(zhì)的表達(dá)量）。 *這是推薦的用量，可以先進(jìn)行常規(guī)的SDS-PAGE和免疫印跡法，確定合適的上樣量。
Q8.	使用哪種蛋白marker？
A8.	不推薦使用蛋白marker。該款產(chǎn)品不需要蛋白marker。因此，建議用來源于大腸桿菌的重組蛋白或者是非磷酸化樣品作為陰性對照代替蛋白marker。
Q9.	此款產(chǎn)品的配離子是什么？
A9.	鋅離子
Q10.	我們怎么知道是因?yàn)榈鞍装l(fā)生磷酸化條帶才會發(fā)生遷移？
A10.	使用12.5% SuperSep™   Ace(Wako 目錄No. 199-14971)進(jìn)行電泳（有相同的膠濃度），檢查目標(biāo)蛋白質(zhì)是否降解。

【參考文獻(xiàn)】

·  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

• 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