| 货号 | 7911C |
| 描述 | The PathScan® Phospho-EGF Receptor (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated EGF receptor protein. An EGF Receptor Rabbit mAb has been coated on the microwells. After incubation with cell lysates, EGF receptor protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Tyrosine Mouse Detection mAb is added to detect captured tyrosine-phosphorylated EGF receptor 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 EGF receptor protein phosphorylated at tyrosine residues. Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | PathScan® Phospho-EGF Receptor (panTyr) Sandwich ELISA Kit detects endogenous levels of tyrosine-phosphorylated EGF receptor protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. 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 | The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10). |
| 存放说明 | 4C |
| 参考文献 | Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4. Hubbard, S.R. et al. (1994) Nature 372, 746-54. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73. |
Figure 1. Treatment of A-431 cells with hEGF #8916 stimulates tyrosine phosphorylation of EGF receptor detected by PathScan® Phospho-EGF Receptor (panTyr) Sandwich ELISA Kit #7911, but does not affect the level of total EGF receptor detected by PathScan® Total EGF Receptor Sandwich ELISA Kit #7250. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using EGF Receptor Antibody #2232 (left panel) or Phospho-EGF Receptor (Tyr1173) (53A2) Rabbit mAb #4407 (right panel) are shown in the bottom figure. 图1. A-431细胞用hEGF #8916处理，刺激EGF受体酪氨酸的磷酸化，用PathScan® Phospho-EGF Receptor (panTyr) Sandwich ELISA Kit #7911进行检测，但不影响用PathScan® Total EGF Receptor Sandwich ELISA Kit #7250检测总EGF受体水平。上图显示在450nm处的吸光度值，而相应的western blots结果见下图，western blots实验所用的抗体为EGF Receptor Antibody #2232 (左面板)或Phospho-EGF Receptor (Tyr1173) (53A2) Rabbit mAb #4407 (右面板)。 | |
Figure 2. The relationship between protein concentration of lysates from untreated and EGF-treated A-431 cells and the absorbance at 450 nm is shown. After starvation, A-431 cells (85% confluence) were treated with hEGF #8916 (100 ng/ml) for 5 min at 37°C and then lysed. 图2. 如图所示，A-431细胞不处理和用EGF处理后，裂解物的蛋白浓度与450 nm处吸光度值的关系。A-431细胞（85%汇合）饥饿处理后，用hEGF #8916 (100 ng/ml)37°C处理5min，随后进行裂解。 |
