PathScan? Phospho-IRS-1 (Ser612) Sandwich ELISA Kit

• 产品编号：CST-7332C      品牌：CST       原厂货号：7332C
• 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 (Ser612) Sandwich ELISA Kit #7332用于检测内源性在612位丝氨酸残基上磷酸化的IRS-1水平。如1图所示，所示为hSkMC和CHO（IR/IRS-1)细胞经胰岛素处理后， Ser612磷酸化的胰岛素受体底物1水平明显提高，所用抗体为Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332。但是Western检测和PathScan® Total IRS-1 Sandwich ELISA Kit #7328检 测（figure1）所示IRS-1（磷酸化和未磷酸化的）总水平没有变化。内部试验证明，该抗体用于检测相应物种的蛋白，但也可能与其它物种的同源蛋白交叉反应。PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit是一种固相三明治酶连免疫吸收试验（ELISA)试剂盒，用于检测内源性612位丝氨酸磷酸化的IRS-1(也称作4PS)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 入鼠源磷酸化IRS-1（Ser612）检测抗体来检测被捕获的磷酸化IRS-1（Ser612）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。Ser612磷酸化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。试剂盒中的抗体是专门定做的。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 (Ser612) Sandwich ELISA Kit #7332 detects endogenous levels of Phospho-IRS-1 when phosphorylated at Ser612. 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. 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). 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 (Ser612) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1 when phosphorylated at Ser612. 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-IRS-1 (Ser612) Mouse Detection Antibody* is added to detect phosphorylation of Ser612 on 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 at Ser612. 
 * 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).