| 货号 | 7242C |
| 描述 | CSTs PathScan® Total Met Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Met protein. A Met Mouse mAb #3127* has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Met proteins are captured by the coated antibody. Following extensive washing, Met Rabbit Antibody #3123* is added to detect both the captured phospho- and nonphospho-Met protein. Anti-rabbit IgG, HRP-linked Antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Met protein. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total Met Sandwich ELISA Kit #7242 detects endogenous levels of total Met protein. As shown in Figure 1, both phospho- and nonphospho-Met proteins from untreated and HGF-treated A431 cell lysates are detected by this kit. In Figure 3, Western blot analysis of protein captured in the Met Mouse mAb #3127 coated microwell shows a single band corresponding to the Met 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 | 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). |
| 存放说明 | 4C |
| 参考文献 | Cooper, C.S. et al. (1984) Nature 311, 29-33. Bottaro, D.P. et al. (1991) Science 251, 802-4. Bardelli, A. et al. (1997) Oncogene 15, 3103-11. Taher, T.E. et al. (2002) J Immunol 169, 3793-800. Schaeper, U. et al. (2000) J Cell Biol 149, 1419-32. Eder, J.P. et al. (2009) Clin Cancer Res 15, 2207-14. Sattler, M. and Salgia, R. (2009) Update Cancer Ther 3, 109-118. |
Figure 1: Non-phospho and phospho Met proteins from untreated and HGF-treated A431 cells can be detected by PathScan® Total Met Sandwich ELISA kit #7242 with similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blots using Met Mouse mAb #3127 (left panel) or Met (Tyr1234/1235) Rabbit mAb #3129 (right panel), are shown in the bottom figure.图1：用PathScan® Total Met Sandwich ELISA kit #7242对经过和未经HGF刺激的A431细胞中磷酸化和非磷酸化的Met蛋白进行检测得到相似的读值。上图显示450nm处的吸光值，下图显示用Met Mouse mAb #3127（右道）或Met (Tyr1234/1235) Rabbit mAb #3129（左道）进行免疫印迹检测的结果。 | |
Figure 2: The relationship between protein concentration of lysates from untreated and HGF-treated A431 cells and kit assay optical density readings is shown. After starvation, A431 cells (85% confluence) were treated with HGF (40 ng/ml) for 5 min at 37°C, and then lysed.图2：经过或未经HGF刺激的A431细胞的裂解液中蛋白含量和吸光值之间的关系。A431细胞在血清饥饿后，用40 ng/ml HGF在37°C.刺激5分钟，然后进行裂解。 | |
Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein is shown. Lysates were prepared from human A431 cells and incubated in wells coated with capture antibody #3127. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. A431 lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using Met antibody #3123. A single band corresponding to the Met protein is detected in the captured material (lane 2).图3：用免疫印迹法检测ELISA微孔中捕获的蛋白显示出ELISA试剂盒的特异性。人A431细胞的裂解液在预包被了捕获抗体#3127的微孔中孵育之后，洗孔，用SDS上样溶液将被捕获的蛋白溶解下来。用Met antibody #3123对最初的A431细胞的裂解液(第1道)以及被捕获的蛋白(第2道)进行免疫印迹检测。被捕获的蛋白在对应Met的位置有唯一条带（第2道）。 |
