| 货号 | 7860C |
| 描述 | The PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-2 when tyrosine phosphorylated. An IRS-2 Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, IRS-2 (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-2 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-2 phosphorylated on tyrosine. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA Kit #7860 detects IRS-2 when tyrosine phosphorylated. As shown in Figure 1, a significant induction of IRS-2 tyrosine phosphorylation can be detected in CHO (IR/4PS) cells following treatment with insulin using the Phospho-IRS-2 (panTyr) Sandwich ELISA Kit #7860. The level of total IRS-2 (phospho and nonphospho) remains unchanged as shown by Western analysis. This kit also detects tyrosine phosphorylated IRS-2 in insulin treated 3T3-L1 adipocytes (data not shown). 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 2 (IRS-2) is one of the major substrates of the insulin receptor kinase (1). In vertebrates, IRS-2 functions as a scaffolding protein to coordinate separate branches of the Insulin/IGF-signaling cascades (2). IRS-2 is essential for normal nutrient homeostasis because it mediates both peripheral insulin action and the effect of IGF-1 on B-cell growth. Mice lacking IRS-2 fail to maintain sufficient compensatory insulin secretion and develop diabetes as young adults (3). |
| 存放说明 | 4C |
| 参考文献 | Sun, X.J. et al. (1991) Nature 352, 73-77. White, M.F. (2002) Am. J. Physiol. Endocrinol. Metab. 283, E413-E422. Withers, D.J. et al. (1998) Nature 391, 900-904. |
Figure 1. Treatment of CHO (IR/4PS) cells with insulin stimulates tyrosine phosphorylation of IRS-2, detected by the PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA Kit #7860, but does not affect the level of total IRS-2 detected by Western analysis. CHO (IR/4PS) cells (80-90% confluent) were starved overnight and treated with 100 nM insulin for 10 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/4PS) cells and incubated in microwells coated with the IRS-2 capture antibody. The wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of CHO (IR/4PS) cell starting lysate (lanes 1, 2, 5 & 6) and the captured protein (lanes 3, 4, 7 & 8) was performed using IRS-2 Antibody #4502 (left panel) and Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 (right panel). The major protein detected in the captured material corresponds to IRS-2 (lanes 3, 4 & 8). 图1，ELISA检测示，用胰岛素处理CHO (IR/4PS)后，会增加IRS-2酪氨酸残基的磷酸化水平(检测试剂盒为PathScan? Phospho-IRS-2 (panTyr) Sandwich ELISA #7861)，但是不会影响IRS-2总蛋白水平(检测试剂盒为PathScan? Total IRS-2 Sandwich ELISA #7884)。稳定转染入胰岛素受体和IRS-2的CHO细胞首先饥饿过夜，然后用100 nM胰岛素于37oC下处理2分钟。上图示450钠米处的随蛋白浓度变化的吸光度值，相应的Western blot实验如下图所示。为了检测酪氨酸残基的磷酸化的IRS-2，首先IRS-2用固化的IRS-2特异性抗体小孔板免疫沉淀分离。然后对小孔进行洗脱并用SDS loading buffer溶解捕获的蛋白。蛋白然后用凝胶电脉进行分离，并用IRS-2 Antibody #4502(左)和Phospho-Tyrosine Mouse mAb 鼠单抗(P-Tyr-100) #9411(右)进行检测。 | |
Figure 2. The relationship between the protein concentration of the lysate from untreated and insulin-treated CHO (IR/4PS) cells and the absorbance at 450 nm is shown. 图2，CHO (IR/4PS)细胞裂解液中蛋白浓度与450钠米处吸光度的相关性，细胞未处理或经胰岛素处理 |
