PathScan? Phospho-Met (panTyr) Sandwich ELISA Kit

• 产品编号：CST-7333C      品牌：CST       原厂货号：7333C
• 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：

CST公司的PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit #7333主要用于检测酪氨酸磷酸化的Met的表达。如图1所示，磷酸化Met高表达于HCC827细胞，该细胞中Met被组成型磷酸化。这种高水平的表达在HCC827细胞裂解液中未加入磷酸酶抑制剂*时被消除。用PathScan® Total Met Sandwich ELISA Kit #7242能够检测到总Met（磷酸化的和未磷酸化的）的量不变（图1）。内部检测显示，本试剂盒能检测列举出的物种蛋白，但不排除用于其它物种同源蛋白检测的可能。*，磷酸酶抑制剂，包括焦磷酸钠、β-甘油磷酸二钠和Na3VO4。CST公司的PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit利用固相双抗夹心酶联免疫检测法对内源性酪氨酸磷酸化的Met的表达进行检测。微孔中已经包被了Met兔抗体，与细胞裂解液共孵育后，Met（磷酸化的和未磷酸化的）能被包被抗体捕获。多次洗板之后，加入磷酸化酪氨酸鼠检测抗体对被捕获的Met蛋白的酪氨酸位点的磷酸化进行检测，然后加入抗鼠IgG, HRP偶联抗体对检测抗体进行识别，最后加入HRP的底物TMB进行显色反应。按照相对光单位，发射光强度与酪氨酸磷酸化的Met蛋白的量成正比。*试剂盒中的抗体为试剂盒特别订制配方。Met作为肝细胞生长因子 (HGF, 亦作扩散因子) 的高亲和性酪氨酸激酶受体是一个二硫键连接的异二聚体，含有一个45 kDa的 α-亚基和一个145 kDa的 β-亚基 (1,2)。α-亚基和β-亚基的氨基端形成胞外区，β-亚基的其余部分跨细胞膜并且含有一个具有激酶活性的胞内区。Met和HGF相互作用会导致多个酪氨酸位点的自磷酸化，继而招募下游信号分子Gab1, c-Cbl和 PI3 K激酶（3）。这一系列基础性事件对于Met激酶相关的所有生物学功能十分重要。胞内Tyr1003位点的磷酸化对于Met的泛素化和降解是必须的（4）。激酶结构域Tyr1234/1235的磷酸化对于Met的激酶活性十分关键。胞内 Tyr1349的磷酸化为Met提供了Gab1 的直接结合位点(5)。在肾、结肠和乳腺癌中发现有Met水平和/或激酶活性的改变，因此Met是肿瘤治疗和诊断的一个靶点（6,7）。

1. Cooper, C.S. et al. (1984) Nature 311, 29-33.
2. Bottaro, D.P. et al. (1991) Science 251, 802-4.
3. Bardelli, A. et al. (1997) Oncogene 15, 3103-11.
4. Taher, T.E. et al. (2002) J Immunol 169, 3793-800.
5. Schaeper, U. et al. (2000) J Cell Biol 149, 1419-32.
6. Eder, J.P. et al. (2009) Clin Cancer Res 15, 2207-14.
7. Sattler, M. and Salgia, R. (2009) Update Cancer Ther 3, 109-118.

原厂资料：

Specificity / Sensitivity

CST's PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit #7333 detects Met when tyrosine phosphorylated. High levels of phospho-Met protein are detected in HCC827 cells where Met is constitutively phosphorylated (Figure 1). These high levels are abolished in HCC827 cells lysed without addition of phosphatase inhibitors* to the lysis buffer. The levels of total Met protein (phospho and nonphospho) as detected by PathScan® Total Met Sandwich ELISA Kit #7242 remain unchanged (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. 
 * Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.

Description

The PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Met when tyrosine phosphorylated. A Met Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, Met (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 Met protein. Anti-mouse IgG, HRP-Linked Antibody 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 Met phosphorylated on tyrosine. 
 Antibodies in kit are custom formulations specific to kit.

Background

Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor) is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl, and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. The addition of a phosphate at cytoplasmic Tyr1003 is essential for Met protein ubiquitination and degradation (4). Phosphorylation at Tyr1234/1235 in the Met kinase domain is critical for kinase activation. Phosphorylation at Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon, and breast. Thus, Met is an attractive cancer therapeutic and diagnostic target (6,7).

1. Cooper, C.S. et al. (1984) Nature 311, 29-33.
2. Bottaro, D.P. et al. (1991) Science 251, 802-4.
3. Bardelli, A. et al. (1997) Oncogene 15, 3103-11.
4. Taher, T.E. et al. (2002) J Immunol 169, 3793-800.
5. Schaeper, U. et al. (2000) J Cell Biol 149, 1419-32.
6. Eder, J.P. et al. (2009) Clin Cancer Res 15, 2207-14.
7. Sattler, M. and Salgia, R. (2009) Update Cancer Ther 3, 109-118.

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).