Translational Control Antibody Sampler Kit

• 产品编号：CST-9918S      品牌：Cell Signaling Technology       原厂货号：9918S
• 产品分类：抗体 > 一抗 > 复合抗体试剂盒
• 应用分类：

其它组分：

描述：

Translational Control Antibody Sampler Kit中每种磷酸化的特异性抗体能够检测仅在指定位点磷酸化的靶蛋白。Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb兔单抗可能与相同位点磷酸化的4E-BP2和4E-BP3蛋白发生交叉反应。通过人工合成人源elF4E蛋白Ser209位点周围相应的磷酸化片段去免疫动物从而制备出磷酸化特异性多克隆抗体。通过蛋白A和多肽亲和层析纯化多克隆抗体。通过人工合成小鼠Akt蛋白Ser473位点，小鼠4E-BP1蛋白Thr37和Thr46位点，人源eIF2α蛋白Ser51位点，人源ribosomal protein S6蛋白Ser235和Ser236，人源p70 S6 kinase Thr389位点周围相应的磷酸化片段分别去免疫动物从而制备出磷酸化特异的兔单克隆抗体。该Translational Control Antibody Sampler Kit提供了一个快速和经济的方式去评估在翻译控制过程中所涉及的多种蛋白。该试剂盒含有足够的一抗和二抗可以进行四次免疫印迹（western blot）实验，包括PI3 kinase和mTOR/FRAP蛋白的特异性抑制剂。在蛋白质翻译控制中关键阶段发生在真核生物起始因子4F (eIF4F)和p70 S6 kinase的调控水平。eIF4F是一个复合物，其功能包括mRNA 5'帽结构的识别。数种刺激例如胰岛素及多种生长和存活因子，主要通过引起一个依赖于PI3K, Akt/PKB和mTOR/FRAP kinases相继激活的信号级联反应来调节eIF4F复合物和p70 S6 kinase。在C-末端Ser473位点磷酸化和活化环上Thr308位点的磷酸化共同激活Akt蛋白，这些修饰是由磷脂依赖的激酶负责的。在体内通过高度选择性抑制剂LY294002使PI3K激酶失活，这可抑制Akt蛋白和这个级联反应的下游元件。通过Akt激活mTOR/FRAP蛋白在Ser2448位点的直接磷酸化是调控它的激酶活性的一个关键调控过程。mTOR/FRAP 蛋白活性能有效的被Rapamycin封闭，从而导致真核生物起始因子4E结合蛋白1 (4E-BP1)的失活以及p70 S6 kinases的激活，其中4E-BP1是翻译起始的抑制因子。由相继的磷酸化促使4E-BP1 蛋白的失活从而引起eIF4E的释放，然而eIF4E与eIF4G和其他因子一起形成一个功能性eIF4F帽子结合复合物。p70 S6 kinases可磷酸化40S核糖体亚基蛋白S6，并且刺激含有5'寡嘧啶特征的 mRNA的翻译。 通过调节eIF4E激酶、Mnk1蛋白和活化的 p70 S6 kinase，Erk通路也涉及在该水平进行调控。Tuberin蛋白是肿瘤抑制因子TSG2基因的一个产物，通过Akt/PKB通路促使该蛋白在Thr1462位点直接磷酸化。Tuberin抑制mammalian target of rapamycin（mTOR），因此这导致p70 S6 激酶的抑制和4E-BP1的激活，从而抑制翻译。

1. Gingras, A. et al. (1999) Annu. Rev. Biochem. 68, 913-963.
2. Gingras, A. C. et al. (2001) Genes and Develop. 15, 807-826.
3. Dennis, P. B. et al. (1999) Curr. Opin. Genet. Dev. 9, 49-54.
4. Volarevic, S. and Thomas, G. (2000) Prog. Nucleic Acid Res. Mol. Biol. 65, 101-127.
5. Pyronnet, S. et al. (2000) Biochem. Pharmacol. 60, 1237-1243.
6. Dever, T.E. (2002) Cell 108, 545-556.
7. Goncharova, E. et al. (2002) J. Biol. Chem. 277, 30958-30967.

原厂资料：

Specificity / Sensitivity

Each phospho-specific antibody in the Translational Control Antibody Sampler Kit detects the intended target only when phosphorylated at the indicated site. Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites.

Source / Purification

Phospho-specific polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser209 of human elF4E. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Phospho-specific rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser473 of mouse Akt, Thr37 and Thr46 of mouse 4E-BP1, Ser51 of human eIF2α, Ser235 and Ser236 of human ribosomal protein S6, and Thr389 of human p70 S6 kinase.

Description

The Translational Control Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in translational control. The kit contains enough primary and secondary antibody to perform four Western blot experiments, as well as specific inhibitors of PI3 kinase and mTOR/FRAP.

Background

Key steps in translational control occur at the level of eukaryotic initiation factor 4F (eIF4F) and p70 S6 kinase regulation. eIF4F is a complex whose functions include the recognition of the mRNA 5' cap structure. Several stimuli, such as insulin and various growth and survival factors, regulate the eIF4F complex and p70 S6 kinase primarily by triggering a signaling cascade dependent on sequential activation of PI3K, Akt/PKB and mTOR/FRAP kinases. Akt is activated by phosphorylation within the C-terminus at Ser473 and within the activation loop at Thr308 by phospholipid-dependent kinases. Inactivation in vivo of PI3K by the highly selective inhibitor LY294002 inhibits Akt and downstream elements of this cascade. Direct phosphorylation of mTOR/FRAP at Ser2448 by Akt is a key regulatory event controlling its kinase activity. mTOR/FRAP activity can be effectively blocked by Rapamycin, leading to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation initiation, and activation of p70 S6 kinases. Inactivation of 4E-BP1 by sequential phosphorylation causes the release of eIF4E, which, together with eIF4G and other factors, forms a functional eIF4F cap binding complex. p70 S6 kinases phosphorylates the 40S ribosomal subunit protein S6 and stimulates the translation of 5' oligopyrimidine tract containing mRNAs. The Erk pathway is also involved in regulation at this level by regulating the eIF4E kinase, Mnk1, and activating p70 S6 kinase. Tuberin, a product of the tumor supressor gene TSG2, is directly phosphorylated at Thr1462 by Akt/PKB. Tuberin inhibits the mammalian target of rapamycin, mTOR, which results in inhibition of p70 S6 kinase and activation of 4E-BP1 and, therefore, inhibition of translation.

1. Gingras, A. et al. (1999) Annu. Rev. Biochem. 68, 913-963.
2. Gingras, A. C. et al. (2001) Genes and Develop. 15, 807-826.
3. Dennis, P. B. et al. (1999) Curr. Opin. Genet. Dev. 9, 49-54.
4. Volarevic, S. and Thomas, G. (2000) Prog. Nucleic Acid Res. Mol. Biol. 65, 101-127.
5. Pyronnet, S. et al. (2000) Biochem. Pharmacol. 60, 1237-1243.
6. Dever, T.E. (2002) Cell 108, 545-556.
7. Goncharova, E. et al. (2002) J. Biol. Chem. 277, 30958-30967.

注意事项：

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not
aliquot the antibodies.