PathScan? Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit

• 产品编号：CST-7063C      品牌：CST       原厂货号：7063C
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
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描述：

PathScan ® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit可以检测Thr389位点磷酸化的内源性的phospho-p70 S6激酶，如图1所示，本试剂盒可以与S6激酶的p85异构体发生交叉反应，试剂盒的灵敏度见图2。 本试剂盒经过内部测试可以检测特定物种的蛋白，但也可能检测其它物种同源性高的蛋白。PathScan ® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit是一种固相三明治酶联免疫吸附方法（ELISA），用来检测在Thr389位点磷酸化的内源性的phospho-p70 S6激酶。兔源p70 S6激酶抗体已经包被在孔中，与细胞的裂解液共孵育后， p70 S6激酶被已包被的抗体捕获。经过彻底的洗涤，加入鼠源phospho-p70 S6 kinase (Thr389) 捕获phospho-p70 S6 kinase (Thr389)蛋白。抗鼠IgG，HRP标记的抗体用来识别结合的抗体。加入HRP底物TMB后产生颜色。颜色的吸光度值与Thr389位点磷酸化的内源性的phospho-p70 S6激酶的含量呈正相关。p70 S6激酶是有丝分裂原活化的丝氨酸/苏氨酸蛋白激酶，是细胞生长和G1期细胞周期正常进行必需的（1,2）。P70 S6激酶可以磷酸化核糖体40S亚基的S6蛋白并参与5‘ oligopyrimidine tract mRNAs的翻译控制（1）。另一个亚型，P85 S6激酶，和p70 S6激酶由相同的基因转录，但氨基端有23个额外的残基，编码核定位信号（1）。两种亚型都处于有丝分裂原激活的PI-3K和rapamycin靶点FRAP/mTOR的信号通路下游，这条通路独立于Ras /MAP激酶级联反应（1）。p70 S6激酶活性取决于在催化、连接和伪底物结构域的多种磷酸化修饰（1）。催化结构域的Thr229和连接结构域的Thr389是调节激酶功能最关键的磷酸化修饰位点（1）。而且与P70激酶体内活性最相关的磷酸化修饰位点是Thr389（3）。Thr389磷酸化可以激活PDK1的Thr229磷酸化（4,5）。这个位点的磷酸化受到各种生长因子，如胰岛素，表皮生长因子和成纤维细胞生长因子，以及血清和一些G蛋白偶联受体配体，wortmannin，LY294002（PI-3K抑制剂）和rapamycin（FRAP/mTOR抑制剂）的刺激（1,6,7）。Ser411，Thr421和Thr424位于伪底物结构域的丝氨酸脯氨酸丰富区（1）。这些位点的磷酸化被认为是通过解除伪底物的抑制进而激活p70 S6激酶（1,2）。另一个对LY294002和rapamycin敏感的磷酸化位点，Ser371，是mTOR的体的底物，与某种抗rapamycin的P70 S6激酶突变体有关（8）。

1. Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82.
2. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
3. Weng, Q.P. et al. (1998) J Biol Chem 273, 16621-9.
4. Pullen, N. et al. (1998) Science 279, 707-10.
5. Alessi, D.R. et al. (1998) Curr Biol 8, 69-81.
6. Polakiewicz, R.D. et al. (1998) J Biol Chem 273, 23534-41.
7. Fingar, D.C. et al. (2002) Genes Dev 16, 1472-87.
8. Saitoh, M. et al. (2002) J Biol Chem 277, 20104-12.

原厂资料：

Specificity / Sensitivity

The PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit detects endogenous levels of p70 S6 kinase when phosphorylated at Thr389, as shown in Figure 1. This kit is predicted to cross-react with the p85 isoform of S6 kinase. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Description

The PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-p70 S6 kinase phosphorylated at Thr389. A p70 S6 kinase rabbit mAb has been coated onto the microwells. After incubation with cell lysates, p70 S6 kinase is captured by the coated antibody. Following extensive washing, a phospho-p70 S6 kinase (Thr389) mouse detection mAb is added to detect the captured phospho-p70 S6 kinase (Thr389). Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. The HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of p70 S6 kinase phosphorylated at Thr389.

Background

p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). A second isoform, p85 S6 kinase, is derived from the same gene and is identical to p70 S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal (1). Both isoforms lie on a mitogen activated signaling pathway downstream of phosphoinositide-3 kinase (PI-3K) and the target of rapamycin, FRAP/mTOR, a pathway distinct from the Ras/MAP kinase cascade (1). The activity of p70 S6 kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains (1). Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function (1). Phosphorylation of Thr389, however, most closely correlates with p70 kinase activity in vivo (3). Prior phosphorylation of Thr389 is required for the action of phosphoinositide 3-dependent protein kinase 1 (PDK1) on Thr229 (4,5). Phosphorylation of this site is stimulated by growth factors such as insulin, EGF and FGF, as well as by serum and some G-protein-coupled receptor ligands, and is blocked by wortmannin, LY294002 (PI-3K inhibitor) and rapamycin (FRAP/mTOR inhibitor) (1,6,7). Ser411, Thr421 and Ser424 lie within a Ser-Pro-rich region located in the pseudosubstrate region (1). Phosphorylation at these sites is thought to activate p70 S6 kinase via relief of pseudosubstrate suppression (1,2). Another LY294002 and rapamycin sensitive phosphorylation site, Ser371, is an in vitro substrate for mTOR and correlates well with the activity of a partially rapamycin resistant mutant p70 S6 kinase (8).

1. Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82.
2. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
3. Weng, Q.P. et al. (1998) J Biol Chem 273, 16621-9.
4. Pullen, N. et al. (1998) Science 279, 707-10.
5. Alessi, D.R. et al. (1998) Curr Biol 8, 69-81.
6. Polakiewicz, R.D. et al. (1998) J Biol Chem 273, 23534-41.
7. Fingar, D.C. et al. (2002) Genes Dev 16, 1472-87.
8. Saitoh, M. et al. (2002) J Biol Chem 277, 20104-12.

注意事项：

* 12 8-well modules -Each module is designed to break apart for 8 tests.
**Kit should be stored at 4°C with the exception of 10X Cell Lysis Buffer, which is stored at –20°C (packaged separately).