| 货号 | 7328C |
| 描述 | The PathScan® Total IRS-1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1. 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, IRS-1 Mouse Detection Antibody* is added to detect 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 absorbance for this developed color is proportional to the quantity of total IRS-1. * 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® Total IRS-1 Sandwich ELISA Kit #7328 detects endogenous levels of IRS-1. As shown in Figure 1, a significant induction of IRS-1 phosphorylation at Ser612 can be detected in hSkMC and CHO (IR/IRS-1) cells following treatment with insulin using the Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332. However, 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 (Figure 1). In Figure 3, Western analysis of protein captured in microwells coated with the IRS-1 antibody shows a major band corresponding to the IRS-1 protein. 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 hSkMC or CHO (IR/IRS-1) cells with insulin stimulates phosphorylation of IRS-1 at Ser612, detected by the PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332, but does not affect the level of total IRS-1 protein detected by PathScan® Total IRS-1 Sandwich ELISA Kit #7328. hSkMC and 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, while the corresponding Western blots, using IRS-1 (L3D12) Mouse mAb #3194 (panels A & B) or Phospho-IRS-1 (Ser612) (L7B8) Mouse mAb #3193 (panels C & D), are shown in the bottom figure. hSkMC和CHO（IR/IRS-1)细胞经胰岛素处理后会激活IRS-1在Ser612位点的磷酸化，所用抗体为Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332。但没有影响IRS-1蛋白的总水平，检测抗体为PathScan® Total IRS-1 Sandwich ELISA Kit #7328。处理方法为：hSkMC和CHO (IR/IRS-1) cells(80-90%)饥饿过液，然后用用100 nM胰岛素于37度下处理7分钟。上图示450nm处的吸光度值，下图示相应的western blots，所用抗体为RS-1 (L3D12) Mouse mAb鼠单抗#3194(A,B)和 Phospho-IRS-1 (Ser612) (L7B8) Mouse mAb鼠单抗#3193（C,D）。 | |
Figure 2. The relationship between the lysate protein concentration from untreated and insulin-treated CHO (IR/IRS-1) cells (A) or hSkMC cells (B) and the absorbance at 450 nm is shown. CHO-IR/IRS-1细胞（A）和hSkMC细胞（B）裂解液蛋白浓度与450nm处的吸光度关系图，细胞未处理或经胰岛素处理。 | |
Figure 3. Kit specificity as demonstrated 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. 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) and the captured protein (lanes 3 & 4) was performed using IRS-1 (L3D12) Mouse mAb #3194. The major band detected in the captured material corresponds to IRS-1 (lanes 3 & 4). 试剂盒特异性：用wetern检测ELISA小孔中捕获的目标蛋白。 CHO (IR/IRS-1)细胞裂解液与小孔中的IRS-1捕获抗体孵育，经洗脱后，捕获蛋白溶解在SDS凝胶上样缓冲液中。western blot检测CHO (IR/IRS-1) 细胞裂解液（1,2）和捕获蛋白（3,4），所用抗体为IRS-1 (L3D12) Mouse mAb鼠单抗 #3194。捕获蛋白检测中的主带对应于IRS-1（3，4）。 |
