- 详细信息
- 询价记录
- 相关实验
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货号: 037-17321 供应商: 上海金畔生物科技有限公司 数量: 大量 英文名: Caspase-6/Caspase-8 Fluorogenic Substrate (Ac-IETD-AMC) 保存条件: -20℃ 规格: 5 mg 肿瘤温馨提示:不可用于临床治疗。
货号: | 037-17321 |
供应商: | 上海金畔生物科技有限公司 |
数量: | 大量 |
英文名: | Caspase-6/Caspase-8 Fluorogenic Substrate (Ac-IETD-AMC) |
保存条件: | -20℃ |
规格: | 5 mg |
产品编号 | 产品名称 | 产品规格 | 产品等级 | 产品价格 |
193-16711 | SuperSep Phos-tag™ (50 μmol/L), 10%, 13 well Phos-tag 13孔10%预制胶 |
5块 | – | – |
190-16721 | SuperSep Phos-tag™ (50 μmol/L), 10%, 17 well Phos-tag 17孔10%预制胶 |
5块 | – | – |
195-16391 | SuperSep Phos-tag™ (50 μmol/L), 12.5%, 13 well Phos-tag 13孔12.5%预制胶 |
5块 | – | – |
193-16571 | SuperSep Phos-tag™ (50 μmol/L), 12.5%, 17 well Phos-tag 17孔12.5%预制胶 |
5块 | – | – |
193-16691 | SuperSep Phos-tag™ (50 μmol/L), 15%, 13 well Phos-tag 13孔15%预制胶 |
5块 | – | – |
196-16701 | SuperSep Phos-tag™ (50 μmol/L), 15%, 17 well Phos-tag 17孔15%预制胶 |
5块 | – | – |
197-16851 | SuperSep Phos-tag™ (50 μmol/L), 17.5%, 13 well Phos-tag 13孔17.5%预制胶 |
5块 | – | – |
194-16861 | SuperSep Phos-tag™ (50 μmol/L), 17.5%, 17 well Phos-tag 17孔17.5%预制胶 |
5块 | – | – |
蛋白磷酸化研究的预制胶
SuperSep Phos-tag™
SuperSep Phos-tag™ 是研究蛋白磷酸化的方法,无需特异性磷酸化抗体或者同位素标记。
◆SuperSep Phos-tag™
Phos-tag™ 是一种预制胶,预先加入了50 μmol/L的Phostag™ Acrylamide,打开包装即可直接使用。预制胶中含有锌作为金属离子,在中心凝胶缓冲液中保存稳定性很好,得到的带条结果也很整齐。
磷酸化蛋白和非磷酸化蛋白作为不同条带分离。
分离后,胶可用于考马斯亮蓝染色,免疫印迹和质谱实验。
已公开的验证蛋白列表,请点击
◆Phos-tag™ SDS-PAGE 的原理
◆在HighWire Search 上搜索到的论文数
◆运用
利用p35的丙氨酸突变体确定Cdk5 激活p35的磷酸化位点
p35常见的磷酸化位点是Ser8和Thr138。但是Ser8和Thr138位点往往会发生丙氨酸突变,产生3种突变体(Ser8突变体:S8A,Thr138突变体:T138A,Ser8和Thr138双突变体:2A)。这3种突变体、野生型p35、Cdk5和没有激酶活性的Cdk5都来源于COS-7细胞。这些细胞裂解液用Phos-tag™ SDS-PAGE和Western blotting 进行检测(检测抗体:p35抗体)。
100 μM Phos-tag ™ 丙烯酰胺, 7.5% 聚丙烯酰胺凝胶
可明确磷酸化位点和条带迁移率的关系!
– 泳道1(条带L2和L4)和泳道5(条带M1):p35在Cdk5的作用下发生了磷酸化;
– 泳道1(条带L2和L4)和泳道3(条带L2和L4):在无激酶活性Cdk5的作用下,大约有一半p35蛋白在Thr138位点
发生磷酸化,同样在138位发生突变的p35蛋白亦是如此。
– 泳道5 (条带M1)和泳道6(条带L3和L4):Ser8和Thr138是主要的磷酸化位点;
– 泳道5(条带M1)、泳道7(条带L1和L2)和泳道8(条带M2):条带M1是Ser8和Thr138都发生磷酸化的条带;
条带M2是只有Ser8磷酸化的条带;条带L1和L2是只有Thr138磷酸化的条带。
※ 条带L1和L3中的X 是不确定哪个位点发生磷酸化的条带;
※ 条带L4是非磷酸化的p35。
【参考文献】
▪ Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag ™ SDS-PAGE. T. Hosokawa, T. Saito, A. Asada, K. Fukunaga, and S. Hisanaga,Mol. Cell. Proteomics, Jun 2010;9: 1133 – 1143.
【结果提供】
理化学研究所 脑科学综合研究中心 回路功能研究核心 记忆功能研究团队 细川智永(Dr. T. Hosokawa)
首都大学东京 理工学研究科 生命科学专业 神经分子功能研究室 久永真市(Dr. S. Hisanaga)
检测含有Dnmt1磷酸化激酶的片段
我们可以确定在片段中含有目的激酶!
① 采用亲和色谱法从鼠脑提取液中纯化GST-Dnmt1(1-290)结合蛋白
② 使用0.3 M 和1 M NaCl 的DNA 纤维素柱洗脱得到目的蛋白
③ GST-Dnmt1(1-290)作为体外激酶实验的反应底物
④ Phos-tag ™ SDS-PAGE 用于Western blotting,确定迁移条带中每个片段的激酶活性
【参考文献】
▪ The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1delta/epsilon. Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and I. Kameshita, Biochem. J.,
【结果提供】
高知大学 综合研究中心 生命、功能物质部门 实验实习机器设施 杉山康宪(Dr. Y. Sugiyama)
香川大学 农学部 应用生物科学科 动物功能生化学研究室 龟下勇(Dr. I. Kameshita)
二维电泳中的应用:分析hnRNP K磷酸化异构体
小鼠巨噬细胞J774.1 经LPS 刺激后,裂解细胞,经过免疫沉淀法分离得到hnRNP K。在二维电泳中,一维是IPG 胶,二维是Phos-tag ™ SDS-PAGE,可分离hnRNP K 的异构体。利用质谱仪,可以确认不同的点代表不同的亚型或修饰蛋白。
同一个等电点的位置上,不同位点发生磷酸化都可以被区分开来
(例: spots 6 vs. 8 and spots 4 vs. 7)
【参考文献】
▪ Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis. Y. Kimura, K. Nagata, N Suzuki, R. Yokoyama, Y. Yamanaka, H. Kitamura, H. Hirano, and O. Ohara, Proteomics, Nov 2010; 10(21): 3884-95.
【结果提供】
横滨市立大学 生命纳米系统科学研究科 生物体超分子系统科学专业 木村弥生(Dr. Y. Kimura)、平野久(Dr. H. Hirano)
理化学研究所RCAI 小原收
◆备注
样品制备:
Phos-tag SDS-PAGE对于蛋白样品中的杂质非常敏感,尤其是螯合剂,钒酸,无机盐,表面活性剂这类物质。
强烈建议在Phos-tag SDS-PAGE之前通过TCA沉淀或渗析法降低杂质含量。
转膜前处理:
另一个必须的步骤是在转膜前,用EDTA去除凝胶中的金属离子(Mn2+或者Zn2+);
该步骤可提高蛋白的转膜效率。
● 分别准备10 mmol/L 含EDTA和不含EDTA 两种1x transfer buffer。
● 将凝胶浸泡在含10 mmol/L EDTA的1x transfer buffer,至少20分钟,温和摇晃。更换新缓冲液,重复3次。
● 将凝胶浸泡在不含10 mmol/L EDTA的1x transfer buffer,10分钟,温和摇晃。
● 转膜操作*。
* 建议用湿法转膜,以提高转膜效率。
◆质量控制
每一批SuperSep Phos-tag™,出厂前均根据其产品规格进行测试,以保证可分离磷酸化和非磷酸化蛋白,以及他们的分离成都在正常参数内。
◆保存温度
2-10℃
◆产品信息
用于Bio-Rad伯乐电泳仪
货号 |
品名 |
电泳仪 |
规格 |
198-17981 |
SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 83×100×3.9mm |
Mini-PROTEAN® Tetra Cell (Bio-Rad Laboratories, Inc.) |
5 块 |
195-17991 |
SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 83×100×3.9mm |
5 块 |
用于Life Technologies电泳仪
货号 |
品名 |
电泳仪 |
规格 |
192-18001 |
SuperSep™ Phos-tag™ (50μmol/L), 7.5%, 17well, 100×100×6.6mm |
XCell SureLock® Mini-Cell (Life Technologies, Inc.) |
5 块 |
199-18011 |
SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 17well, 100×100×6.6mm |
5 块 |
用于Wako EasySeperator电泳仪
产品编号 | 产品 | 凝胶浓度 | 孔数 | 包装 |
192-17401 | SuperSep™ Phos-tag™ (50 μmol/L) Phos-tag™ 预制胶50 μmol/L |
6.0% | 13孔 | 5块 |
199-17391 | 6.0% | 17孔 | 5块 | |
195-17371 | 7.5% | 13孔 | 5块 | |
192-17381 | 7.5% | 17孔 | 5块 | |
193-16711 | 10.0% | 13孔 | 5块 | |
190-16721 | 10.0% | 17孔 | 5块 | |
195-16391 | 12.5% | 13孔 | 5块 | |
193-16571 | 12.5% | 17孔 | 5块 | |
193-16691 | 15.0% | 13孔 | 5块 | |
196-16701 | 15.0% | 17孔 | 5块 | |
197-16851 | 17.5% | 13孔 | 5块 | |
194-16861 | 17.5% | 17孔 | 5块 |
◆相关产品
产品编号 | 产品名称 | 规格 |
058-07681 | EasySeparator Phos-tag预制凝胶的配套电泳槽 |
1 set |
Phos-tag™ 系列
磷酸化蛋白新方法!
Phos-tag™是一种能与磷酸离子特异性结合的功能性分子。它可用于磷酸化蛋白的分离(Phos-tag™ Acrylamide)、Western Blot检测(Phos-tag™ Biotin)、蛋白纯化 (Phos-tag™Agarose)及质谱分析MALDI-TOF/MS (Phos-tag™ Mass Analytical Kit)。
◆Phos-tag™ 的基本结构:
特点:
与-2价磷酸根离子的亲和性和选择性高于其它阴离子
在pH 5-8的生理环境下生成稳定的复合物
◆原理:
◆相关应用:
◆相关产品:
产品名称 |
用 途 |
Phos-tag™ Acrylamide |
分离: SDS – PAGE 分离不同磷酸化水平的蛋白 |
SuperSep Phos-tag™ |
分离: 预制胶中含有50μM Phos-tag™ Acrylamide |
Phos-tag™ Biotin |
检测: 代替 Western Blot 检测中的磷酸化抗体 |
Phos-tag™ Agarose |
纯化: 通用柱层析,纯化磷酸化蛋白 |
Phos-tag™ Mass Analytical Kit |
分析: 用于质谱 MALDI-TOF/MS 分析,提高磷酸化分子的检测灵敏度 |
phos-tag™由日本广岛大学研究生院医齿药学综合研究科医药分子功能科学研究室开发。
更多产品信息,请点击:http://phos-tag.jp
Phos-tag 第5版说明书
Phos-tag系列 ver 5
Q1. |
我们可以采用哪种凝胶染色法? |
A1. |
所有的染色法都可使用,最常用的例如考马斯亮蓝染色法,负染,银染和荧光染色等。 |
Q2. |
用考马斯亮蓝染法染色,着色不明显。 |
A2. |
在微波炉内进行脱色,会取得比较满意的效果。 方法:将染色的胶放在100毫升去离子水里,用擦拭纸包裹胶,再放进微波炉加热几分钟后更换去离子水,并重复上述步骤3或4次。请注意防止盛放胶的容器温度过高。 |
Q3. |
此款产品能否用于免疫印迹? |
A3. |
可以,如果用EDTA清除胶里面含有的锌,可以提高转膜的效率。 方法:胶浸在含有10 mmol/L EDTA的转移缓冲液(25 mmol/L tris、192 mmol/L甘氨酸,10%甲醇)里轻轻搅拌10分钟。重复上述步骤3次。然后放进不含EDTA的转移缓冲液(25 mmol/L tris、192 mmol/L的甘氨酸,10%甲醇)里搅拌10分钟并转移到PVDF膜或NC膜(硝酸纤维素膜)上。 |
Q4. |
条带扭曲了。 |
A4. |
含有EDTA,无机盐,表面活性剂等的样品可能会导致条带弯曲或者拖尾。通过TCA或透析沉淀脱盐样品。空白泳道也会导致相邻样品条带弯曲,在空白泳道加与样品相同体积的样品缓冲液(x1)。 |
Q5. |
磷酸化蛋白和非磷酸化蛋白不能分离。 |
A5. |
将β–酪蛋白(038-23221)作为Phos-tag SDS-PAGE电泳的阳性对照,将用碱性磷酸酶处理的β–酪蛋白作为阴性对照,并检查条带的迁移。如果只有一个条带,可能是由于Phos-tag™或丙烯酰胺的浓度没有优化导致磷酸化和非磷酸化蛋白不能分离。 |
Q6. |
可用在细胞的粗提物上吗? |
A6. |
可以的,可能Rf值可能有稍低,由于目的蛋白的因素,条带可能会比较模糊。 |
Q7. |
上样量多少? |
A7. |
1至5微克纯化的蛋白(考马斯亮蓝染法),10至30微克的组织或细胞提取物(取决于蛋白质的表达量)。 *这是推荐的用量,可以先进行常规的SDS-PAGE和免疫印迹法,确定合适的上样量。 |
Q8. |
使用哪种蛋白marker? |
A8. |
不推荐使用蛋白marker。该款产品不需要蛋白marker。因此,建议用来源于大肠杆菌的重组蛋白或者是非磷酸化样品作为阴性对照代替蛋白marker。 |
Q9. |
此款产品的配离子是什么? |
A9. |
锌离子 |
Q10. |
我们怎么知道是因为蛋白发生磷酸化条带才会发生迁移? |
A10. |
使用12.5% SuperSep™ Ace(Wako 目录No. 199-14971)进行电泳(有相同的胶浓度),检查目标蛋白质是否降解。 |
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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.
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· 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.
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· 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.
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· Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics[J]. Proteomics, 2016,Yang W, Jackson B, Zhang H.
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· 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,Fojtikova V, Bartosova M, Man P, et al.
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· 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.
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· 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.
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· 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.
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· Fine tuning chloroplast movements through physical interactions between phototropins[J]. Journal of Experimental Botany, 2016: erw265,Sztatelman O, Łabuz J, Hermanowicz P, et al.
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· 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.
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· 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.
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References on Phos-tag™ Chemistry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule, Rapid Communications of Mass Spectrometry, 17, 2075-2081 (2003), H. Takeda, A. Kawasaki, M. Takahashi, A. Yamada, and T. Koike
Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc (II) complex, Dalton Transactions, 1189-1193 (2004), E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro, and T. Koike
Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule, Journal of Lipid Research, 45, 2145-2150 (2004), T. Tanaka, H. Tsutsui, K. Hirano, T. Koike, A. Tokumura, and K. Satouchi
Production of 1,2-Didocosahexaenoyl Phosphatidylcholine by Bonito Muscle Lysophosphatidylcholine/Transacylase, Journal of Biochemistry,136, 477-483 (2004), K. Hirano, H. Matsui, T. Tanaka, F. Matsuura, K. Satouchi, and T. Koike
Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins, Journal of Separation Science, 28, 155-162 (2005), E. Kinoshita, A. Yamada, H. Takeda, E. Kinoshita-Kikuta, and T. Koike
Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule, Analytical Chemistry, 77, 3979-3985 (2005), K. Inamori, M. Kyo, Y. Nishiya, Y. Inoue, T. Sonoda, E. Kinoshita, T. Koike, and Y. Katayama
Phosphate-binding tag: A new tool to visualize phosphorylated proteins, Molecular & Cellular Proteomics, 5, 749-757 (2006), E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, and T. Koike
Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH, Proteomics, 6, 5088-5095 (2006), E. Kinoshita-Kikuta, E. Kinoshita, A. Yamada, M. Endo, and T. Koike
Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 360, 160-162 (2007), S. Yamada, H. Nakamura, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and Y. Shiro
Label-free kinase profiling using phosphate-affinity polyacrylamide gel electrophresis, Molecular & Cellular Proteomics, 6, 356-366 (2007), E. Kinoshita-Kikuta, Y. Aoki, E. Kinoshita, and T. Koike
A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike (The phosphate group at DNA-terminal is efficiently captured by Zn2+.Phos-tag.)
Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule, Journal of Biomolecular Techniques, 18, 278-286 (2007), T. Nakanishi, E. Ando, M. Furuta, E. Kinoshita, E. Kikuta-Kinoshita, T. Koike, S. Tsunasawa, and O. Nishimura
A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate- affinity SDS-PAGE, Proteomics, 8, 2994-3003 (2008), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, S. Yamada, H. Nakamura, Y. Shiro, Y. Aoki, K. Okita, and T. Koike
FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway, Nature Structural & Molecular Biology, 15, 1138-1146 (2008), M. Ishiai, H. Kitao, A. Smogorzewska, J. Tomida, A. Kinomura, E. Uchida, A. Saberi, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, S. Tashiro, S. J. Elledge, and M. Takata
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Two-dimensional phosphate affinity gel electrophoresis for the analysis of phosphoprotein isotypes , Electrophoresis, 30, 550-559 (2009), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, Y. Aoki, S. Ohie, Y. Mouri, and T. Koike
Formation of lysophosphatidic acid, a wound-healing lipid, during digestion of cabbage leaves, Bioscience, Biotechnology, and Biochemistry,73, 1293-300 (2009), T. Tanaka, G. Horiuchi, M. Matsuoka, K. Hirano, A. Tokumura, T. Koike, and K. Satouchi
A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides, Analytical Biochemistry, 388, 235-241, (2009), K. Takiyama, E. Kinoshita, E. Kinoshita-Kikuta, Y. Fujioka, Y. Kubo, and T. Koike
Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates, Analytical Biochemistry, 389, 83-85, (2009), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose, Proteomics, 9, 4098- 4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Ujihara, and T. Koike
Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE, Nature Protocols, 4, 1513-1521 (2009), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag, Rapid Communications in Mass Spectrometry, 24, 1075-1084 (2010), J. Morishige, M. Urikura, H. Takagi, K. Hirano, T. Koike, T. Tanaka, and K. Satouchi
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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
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产品编号 | 产品名称 | 产品规格 | 产品等级 | 产品价格 |
141-08701 | Nafamostat Mesylate 甲磺酸萘莫司他 | 100mg | - | - |
078-03381 | Gabexate Mesilate 加贝酯 | 1g | - | - |
抗凝血酶药成分
◆甲磺酸萘莫司他(Nafamostat Mesylate)
CAS No. 82956-11-4 C19H17N5O2・2CH3SO3H=539.58 纯度:98.0+%(HPLC) 可溶性溶解:水 用途(作用):合成凝血酶剂。抑制丝氨酸蛋白酶从而抑制血液凝固。 |
◆加贝酯(Gabexate Mesilate)
CAS No. 56974-61-9 C17H27N3O7S=417.48 纯度:98.0+%(HPLC) 可溶性溶解:水 用途(作用):合成凝血酶剂。抑制丝氨酸蛋白酶从而抑制血液凝固。 |
相关资料详情请查看:http:///pdf/show/80.html
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磷酸酶(碱性)(大肠杆菌)
英文名:Phosphatase (Alkaline) (E. coli)
货号:E-ALPEC
规格:400 Units
High purity recombinant Alkaline phosphatase (E. coli) for use in research, biochemical enzyme assays and in vitrodiagnostic analysis.
EC 3.1.3.1
Orthophosphoric-monoester phosphohydrolase (alkaline optimum).
Recombinant from E. coli.
In 3.2 M ammonium sulphate containing 1 mM MgCl2 and 0.1 mM ZnSO4.
Specific activity: ~ 10 U/mg (37oC, pH 10.4, 4-Nitrophenyl phosphate).
Stable at 4oC for > 2 years.
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抗原修复就是利用虎穴试剂盒热的作用将抗原重新暴露出现或修正过来的过程。
常用抗原修复法介绍:
1. 真空负压抗原修复法:
① 切片脱蜡至水。
②0.3%H2O2甲醇真空负压处理5分钟。
③自来水洗,蒸馏水洗。
④0.01M柠檬酸盐缓冲液(PH6.0),真空负压干燥箱预先调至95℃,真空负压处理10分钟。
⑤待修复注降至室温的一,PBS洗3次,随后按选好的年免疫组化染色方法进行染色。这是一种操作简单,效果特佳,温度恒定,能一次性处理大量切片的方法。
2.微波辐射抗原修复法:
①切片脱蜡至水。
②0.3%H2O2甲醇处理10分钟。
③自来水洗,蒸馏水洗。
④ 0.01M柠檬酸盐缓冲液(PH6.0),于微波炉内微波辐射10分钟,如检测Er和Pr则需要20辐射分钟左右。
⑤待修复液降至室温后,PBS洗3次,随后按选好的免疫组织化学的染色方法进行染色。
3.高压抗原修复法:
①切片脱蜡至水。
②0.3%H2O2甲醇处理切片10分钟。
③自来水洗,蒸馏水洗。
④切片放入抗原修复液中,边同容器放入高压锅中加热至沸腾。盖上压力阀至喷汽后持续1-4分钟。
⑤待修复液恢复至室温后,PBS洗3次,随后按选好的免疫组织化学染色方法进行染色。
4.隔水热抗原修复法:
①切片脱蜡至水。
②0.3%H2O2甲醇处理切片10分钟。
③自来水洗,蒸馏水洗。
④切片放入0.01M柠檬酸盐缓冲液(PH6.0)中,边同容器一起放入水溶锅中加热,其间应不断用温度计测其温度,待抗原修复液的温度达到有效温度后(92℃↑)。即开始计时,持续40分钟。⑤待抗原修复液恢复至室温后,PBS洗3次,随后按选定好的免疫组化染色方法进行染色。
5.电炉加热抗原修复法:
①切片脱蜡至水。
②0.3%H2O2甲醇处理切片10分钟。
③自来水洗,蒸馏水洗。
④ 将切片放入抗原修复液中于电炉上加热,不时用温度计测量温度,当达92℃后,即可拔离电源,当温度低于92℃时,再插上电源,如此反复持续至10分钟左右。
⑤待抗原修复液降至室温后,PBS洗3次,随后按选定的免疫组化染色方法进行染色。对各种抗原修复方法的评价。
抗原修复的方法选择:
抗原修复关系到组织抗原能否暴露充分,这对免疫组化染色效果有很大影响。不适当的抗原修复方式会导致染色弱阳性。因此应当根据不同的抗原特点,采用不同的修复方法。不适当的抗原修复方式会导致染色弱阳性。因此应当根据不同的抗原特点,采用不同的修复方法。对某些细胞内抗原(如Fas、Bax、FⅧ等)和细胞间质抗原(如Laminin、CoIV等)则需要用酶消化法处理切片。用于IHC消化的酶很多,所选酶的种类,使用的浓度,PH值,消化时间及温度等均要视组织固定的不同,所检测抗原的性质、组织类型的不同而异,应通过预实验摸索定。使用酶消化的原则:胰蛋白酶和蛋白酶K一般用于细胞内抗原,如Keratin,CEA,GFAP等。一般说来,胰蛋白酶消化能力较胃蛋白酶弱,主要用于细胞内抗原的消化,胃蛋白酶主要用于细胞间抗原的消化,如fibronectin,Laminin,各型胶原等。消化时间和组织固定的长短呈正比。在用酶消化,热修复后均不能获得结果的前提下使用抗原修复与酶消化结合的方法,有时会取得较为满意的结果。一般蛋白酶K和微波相结合,但应注意,可能检测的抗原无论何种性质均会在核内出现假阳性反应。以高压加热方法、微波加热方法较为稳定,?高压加热方法效果优于微波加热方法和单纯加热方法。溶液的浓度对修复效果无任何影响,而PH值则影响较大。缓冲液以柠檬酸缓冲液和Tris-HCL较常用。
免疫组化抗原修复法的注意事项:
1、pH的应用范围及选择抗原修复液的pH非常重要,有效的抗原修复pH要比修复液的化学成分更重要,同样的修复液随着pH的升高染色的强度逐渐增强,但最佳pH范围为6.0-10.0。目前大家公认的最好的抗原修复液是pH6.0的柠檬酸盐缓冲液和pH8.0的EDTA缓冲液。作为通用修复液碱性pH的修复液要比酸性的有效,而对固定很长时间旧的存档组织,酸性pH的修复液则优于碱性的修复液。
2、抗原修复时应选择最佳温度70-90℃的温度对未经固定的蛋白质可发生变性,但经福尔马林固定的蛋白质,温度必须达到92℃以上方能使其变性。研究结果显示,温度为92-98℃是合适的,95℃效果最好。
3、抗原修复液必须遵循自然降温规律,否则效果不好或达不到抗原修复的目的抗原修复持续时间过后,取出放于室温中让其慢慢地降温,绝对不能为了争取时间,强行用冰块或冷水使其降温。这是因为当高温中的抗原蛋白分子链脱离了其他的束缚或联结,要有一个自然环境让其自然放松下来,随着温度的降低,它们会慢慢地恢复原来的形态和构型。
4、尽量使用足量的抗原修复液应用于抗原修复的液体,一定要充足,防止切片干涸。应用大量的抗原修复液时,由于它的量较大,可延缓液体的沸腾时间,增加切片受微波辐射的量,对抗原修复将达到彻底。
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