PathScan? Phospho-IRS-1 (panTyr) Sandwich ELISA Kit

• 产品编号：CST-7133C      品牌：CST       原厂货号：7133C
• 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-IRS-1 (panTyr) Sandwich ELISA Kit #7133用于检测酪氨酸残基磷酸化的IRS-1蛋白。如图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western blot检测表明IRS-1总蛋白水平未发生变化。该试剂盒同样可以检测胰岛素处理的3T3-L1脂肪细胞和MCF7中酪氨酸磷酸化的 IRS-1水平(数据未展示)。内部测试证明，该试剂盒除了可以检测相应物种的对应蛋白外，也可与其它物种的同源蛋白交互反应。PathScan? Phospho-IRS-1 (panTyr) Sandwich ELISA是一种固相三明治酶联免疫试验(ELISA)试剂盒，用于检测内源性酪氨酸磷酸化的IRS-1(也称作4PS)总蛋白。步骤如下：首先在微孔中结合有固相的兔抗IRS-1抗体，然后将细胞裂解液于固相抗体中孵育，IRS-1(磷酸化与非磷酸化的)蛋白被固相抗体所捕获。经过洗脱后，非特异性结合的成分被洗脱下来，然后加入磷酸化的鼠源IRS-1检测抗体来检测被捕获的酪氨酸磷酸化IRS-1蛋白，然后再加入辣根过氧化物酶耦联的抗体来检测特异性结全的检测抗体。最后加入辣根过氧化物酶的底物TMB来进行显色。酪氨酸磷酸化的IRS-1的浓度与显色的强度成正比，因此可以借此对IRS-1定量。该试剂盒中的抗体是根据试剂盒定制的。胰岛素受体底物1(IRS-1)是胰岛素受体激酶的一类主要底物(1)。 IRS-1包含多个酪氨酸磷酸化位点，可以作为含SH2结构域蛋白的停靠位点，这些蛋白调控着胰岛素的代谢和促生长功能(2-4)　。IRS-1还包含着起过30个潜在的丝氨酸/苏氨酸磷酸化位点，307位丝氨酸可以被JNK(5)和IKK(6)磷酸化，789位的丝氨酸可以被SIK-2（是AMPK家族成员）磷酸化(7)。PKC和mTOR途径可以分别磷酸化IRS-1的612位丝氨酸和636/639位丝氨酸(8,9)。PKCθ可以介导IRS-2在1101位丝氨酸位的磷酸化，此位点的磷酸化会导致细胞胰岛素信号通路的抑制，因此它可能在某些肥胖模型的胰岛素抵抗中起着某些作用(10)。

1. Sun, X.J. et al. (1991) Nature 352, 73-77.
2. Sun, X.J. et al. (1992) J. Biol. Chem. 267, 22662-22672.
3. Myers Jr., M.G. et al. (1993) Endocrinology 132, 1421-1430.
4. Wang, L.M. et al. (1993) Science 261, 1591-1594.
5. Rui, L. et al. (1997) J. Clin. Invest. 107, 181-189.
6. Gao, Z. et al. (2002) J. Biol. Chem. 277, 48115-48121.
7. Horike, N. et al. (2003) J. Biol. Chem. 278, 18440-18447.
8. Ozes, O.N. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4640-4645.
9. De Fea, K. and Ruth, R.A. (1997) Biochemistry 36, 12939-12947.
10. Li, Y. et al. (2004) J. Biol. Chem. 279, 45304-45307.

原厂资料：

Specificity / Sensitivity

CST''s 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.

Description

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.

Background

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).

1. Sun, X.J. et al. (1991) Nature 352, 73-77.
2. Sun, X.J. et al. (1992) J. Biol. Chem. 267, 22662-22672.
3. Myers Jr., M.G. et al. (1993) Endocrinology 132, 1421-1430.
4. Wang, L.M. et al. (1993) Science 261, 1591-1594.
5. Rui, L. et al. (1997) J. Clin. Invest. 107, 181-189.
6. Gao, Z. et al. (2002) J. Biol. Chem. 277, 48115-48121.
7. Horike, N. et al. (2003) J. Biol. Chem. 278, 18440-18447.
8. Ozes, O.N. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4640-4645.
9. De Fea, K. and Ruth, R.A. (1997) Biochemistry 36, 12939-12947.
10. Li, Y. et al. (2004) J. Biol. Chem. 279, 45304-45307.

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).