| 货号 | 7038C |
| 描述 | The PathScan® Total p70 S6 Kinase Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p70 S6 kinase. A p70 S6 kinase rabbit antibody has been coated onto the microwells. After incubation with cell lysates, p70 S6 kinase is captured by the coated antibody. Following extensive washing, a biotinylated p70 S6 kinase rabbit detection mAb is added to detect the captured p70 S6 kinase. HRP-linked streptavidin 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 present. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | The PathScan® Total p70 S6 Kinase Sandwich ELISA Kit detects endogenous levels of p70 S6 kinase, 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. |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | 4C |
| 参考文献 | Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9. Weng, Q.P. et al. (1998) J Biol Chem 273, 16621-9. Pullen, N. et al. (1998) Science 279, 707-10. Alessi, D.R. et al. (1998) Curr Biol 8, 69-81. Polakiewicz, R.D. et al. (1998) J Biol Chem 273, 23534-41. Fingar, D.C. et al. (2002) Genes Dev 16, 1472-87. Saitoh, M. et al. (2002) J Biol Chem 277, 20104-12. |
Figure 1. Treatment of MCF7 cells with IGF-1 stimulates phosphorylation of p70 S6 kinase at Thr389 as detected by the PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063, but does not affect the level of total p70 S6 kinase as detected by PathScan® Total p70 S6 Kinase Sandwich ELISA Kit #7038. MCF7 cells were treated with 100 ng/ml IGF-1 #3093 for 20 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while corresponding western blots using p70 S6 Kinase Antibody #9202 (left panel) and Phospho-p70 S6 Kinase (Thr389) (1A5) Mouse mAb #9206 (right panel) are shown in the bottom figure. Figure 1，采用IGF-1处理MCF7细胞，使得p70 S6激酶在Thr389位点发生磷酸化，检测用试剂盒为PathScan ® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063，但不影响总p70 S6激酶的水平，检测用PathScan ® Total p70 S6 Kinase Sandwich ELISA Kit #7038. 。 MCF-7细胞采用100 ng/ml IGF-1 #3093在37度处理20分钟。在450nm处读取吸光度值（上图），相对应western blot采用抗体为 p70 S6 Kinase Antibody #9202 (左泳道) 和Phospho-p70 S6 Kinase (Thr389) (1A5) Mouse mAb鼠单抗 #9206 (右泳道），见下图。 | |
Figure 2. The relationship between lysate protein concentration from untreated and IGF-1-treated MCF7 cells and the absorbance at 450 nm using the PathScan® Total p70 S6 Kinase Sandwich ELISA Kit #7038 is shown. MCF7 cells were treated with 100 ng/ml IGF-1 #3093 for 20 minutes at 37ºC and then lysed. Firure2，采用IGF-1处理MCF7细胞和未处理组细胞提取物中蛋白浓度与450nm处吸光度值的关系图，采用的试剂盒为PathScan ® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063 。 MCF-7细胞采用100 ng/ml IGF-1 #3093在37度处理20分钟，然后裂解。 |
