| 货号 | 7133C |
| 描述 | The PathScan® Phospho-IRS-1 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1 when tyrosine phosphorylated. An IRS-1 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, IRS-1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Tyrosine Mouse Detection Antibody is added to detect tyrosine phosphorylation of the captured IRS-1 protein. Anti-mouse IgG, HRP-linked Antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of IRS-1 phosphorylated on tyrosine. Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Mouse/Rat |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Phospho-IRS-1 (panTyr) Sandwich ELISA Kit #7133 detects IRS-1 when tyrosine phosphorylated. As shown in Figure 1, a significant induction of IRS-1 tyrosine phosphorylation can be detected in CHO (IR/IRS-1) cells following treatment with insulin using the Phospho-IRS-1 (panTyr) Sandwich ELISA Kit #7133. The level of total IRS-1 (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total IRS-1 Sandwich ELISA Kit #7328. This kit also detects tyrosine phosphorylated IRS-1 in insulin treated MCF7 cells and 3T3-L1 adipocytes. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. |
| 供应商 | CST |
| 背景 | Insulin receptor substrate 1 (IRS-1) is one of the major substrates of the insulin receptor kinase (1). IRS-1 contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2-domain containing proteins that mediate the metabolic and growth-promoting functions of insulin (2-4). IRS-1 also contains over 30 potential serine/threonine phosphorylation sites. Ser307 of IRS-1 is phosphorylated by JNK (5) and IKK (6) while Ser789 is phosphorylated by SIK-2, a member of the AMPK family (7). The PKC and mTOR pathways mediate phosphorylation of IRS-1 at Ser612 and Ser636/639, respectively (8,9). Phosphorylation of IRS-1 at Ser1101 is mediated by PKCθ and results in an inhibition of insulin signaling in the cell, suggesting a potential mechanism for insulin resistance in some models of obesity (10). |
| 存放说明 | 4C |
| 参考文献 | Sun, X.J. et al. (1991) Nature 352, 73-77. Sun, X.J. et al. (1992) J. Biol. Chem. 267, 22662-22672. Myers Jr., M.G. et al. (1993) Endocrinology 132, 1421-1430. Wang, L.M. et al. (1993) Science 261, 1591-1594. Rui, L. et al. (1997) J. Clin. Invest. 107, 181-189. Gao, Z. et al. (2002) J. Biol. Chem. 277, 48115-48121. Horike, N. et al. (2003) J. Biol. Chem. 278, 18440-18447. Ozes, O.N. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4640-4645. De Fea, K. and Ruth, R.A. (1997) Biochemistry 36, 12939-12947. Li, Y. et al. (2004) J. Biol. Chem. 279, 45304-45307. |
Figure 1. Treatment of CHO (IR/IRS-1) cells with insulin stimulates tyrosine phosphorylation of IRS-1, detected by the PathScan® Phospho-IRS-1 (panTyr) Sandwich ELISA Kit #7133, but does not affect the level of total IRS-1 detected by PathScan® Total IRS-1 Sandwich ELISA Kit #7328. CHO (IR/IRS-1) cells (80-90% confluent) were starved overnight and treated with 100 nM insulin for 7 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure. Kit specificity is demonstrated in the bottom figure by Western analysis of the ELISA microwell captured protein. Lysates were prepared from CHO (IR/IRS-1) cells and incubated in microwells coated with the IRS-1 capture antibody. The wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of CHO (IR/IRS-1) cell starting lysate (lanes 1, 2, 5 & 6) and the captured protein (lanes 3, 4, 7 & 8) was performed using IRS-1 (L3D12) Mouse mAb #3194 (left panel) and Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 (right panel). The major protein detected in the captured material corresponds to IRS-1 (lanes 3, 4 & 8). 图1所示，用胰岛素对CHO (IR/4PS)细胞进行处理，IRS-1酪氨酸磷酸化水平被显著激活(检测试剂盒为Phospho-IRS-1 (panTyr) Sandwich ELISA Kit #7133)，但是PathScan® Total IRS-1 Sandwich ELISA Kit #7328检测表明IRS-1总蛋白水平未发生变化。CHO (IR/IRS-1)细胞（80－90%）饥饿后，用100 nM胰岛素于37度下处理7分钟，上图示450nm处的吸光度值。下图示用western检测ELISA试剂盒中捕获蛋白，来显示试剂盒的灵敏性。将96孔洗脱后，捕获蛋白溶解在SDS gel loading buffer中。Western检测分析CHO (IR/IRS-1)细胞裂解液（lanes 1, 2, 5 & 6）和Elisa捕获蛋白（lanes 3, 4, 7 & 8），所用抗体为IRS-1 (L3D12) Mouse mAb鼠单抗 #3194 (left panel) 和Phospho-Tyrosine Mouse mAb 鼠单抗(P-Tyr-100) #9411 (right panel)。R捕获蛋白中的主带对应于IRS-1(lanes 3, 4 & 8). | |
Figure 2. The relationship between the protein concentration of the lysate from untreated and insulin-treated CHO (IR/IRS-1) cells and the absorbance at 450 nm is shown. CHO (IR/IRS-1)细胞裂解液目标蛋白浓度与450nm处吸光度值关系图，细胞未处理或用胰岛素处理。 |
