PathScan? Phospho-EGF Receptor (Tyr1068) Chemiluminescent Sandwich ELISA Kit

• 产品编号：CST-7036C      品牌：CST       原厂货号：7036C
• 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®磷酸化- EGF Receptor (Tyr1068)双抗夹心法化学发光检测试剂盒#7036主要用于检测人类细胞中磷酸化的EGFR(Tyr1068)蛋白的表达。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。CST公司的PathScan®磷酸化-EGFR(Tyr1068)双抗夹心法化学发光检测试剂盒利用固相双抗夹心酶联免疫检测法并结合化学发光读取系统，对内源性磷酸化的EGFR(Tyr1068)蛋白的表达进行检测。化学发光ELISA检测体系比传统的发色检测体现出更广的动态范围和更高的灵敏度。本化学发光ELISA检测在半量微孔板中进行，并在微量样本检测中呈现出更强的信号和检测灵敏度。微孔中已经包被了抗EGFR的小鼠源单克隆抗体，与细胞裂解液共孵育后，磷酸化的和未磷酸化的EGFR均能被包被抗体捕获。多次洗板之后，加入抗磷酸化-EGFR(Tyr1068)的兔源抗体对被捕获的磷酸化-EGFR(Tyr1068)蛋白进行检测，然后加入HRP标记的抗兔的IgG#7074对检测抗体进行识别，最后加入化学发光检测试剂检测信号。按照相对光单位，发射光强度与磷酸化-EGFR(Tyr1068）蛋白的量成正比。试剂盒中的抗体为试剂盒特别订制配方。表皮生长因子（EGF）受体是一个属于HER/ErbB蛋白家族的跨膜酪氨酸激酶。配体的结合导致了受体的二聚化，自磷酸化，下游信号分子的激活，内化以及溶酶体降解 (1,2)。表皮生长因子受体（EGFR）在激酶域Tyr845位点的磷酸化与稳定激活回路有关，并且维持了酶的激活状态，为底物蛋白提供了可结合的表位(3,4)。c-Src与 EGFR 在 Tyr845位点的磷酸化有关(5)。PLCγ的SH2结构域结合到磷酸化的Tyr992位点，将引起PLCγ介导的下游信号通路的激活(6)。EGFR Tyr1045位点的磷酸化则为c-Cbl生成一个主要的停靠位点，这个衔接蛋白将导致受体的泛素化以及EGFR活化后的降解(7,8)。GRB2衔接蛋白则结合到活化后的EGFR磷酸化的Tyr1068位点(9)。一对磷酸化EGFR残基(Tyr1148 and Tyr1173)为Shc支架蛋白提供了停靠点，这两个位点都与MAPK激酶通路的激活有关(2)。EGFR在特定的丝氨酸和苏氨酸残基上的磷酸化减弱了EGFR激酶的活性。EGFR羧基末端残基Ser1046 和 Ser1047被CaM 激酶II磷酸化，这两个丝氨酸任何一个突变都将导致EGFR酪氨酸自身磷酸化的上调(10)。

1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.
2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.
3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.
4. Hubbard, S.R. et al. (1994) Nature 372, 746-54.
5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.
6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.
9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

原厂资料：

Specificity / Sensitivity

PathScan® Phospho-EGF Receptor (Tyr1068) Chemiluminescent Sandwich ELISA Kit #7036 detects endogenous levels of phospho-EGF Receptor (Tyr1068) protein in human cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Description

CST's PathScan® Phospho-EGF Receptor (Tyr1068) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-EGF Receptor (Tyr1068) protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. An EGF Receptor Mouse mAb has been coated onto the microwells. After incubation with cell lysates, EGF receptor proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, Phospho-EGF Receptor (Tyr1068) Rabbit mAb is added to detect the captured phospho-EGF Receptor protein. Anti-rabbit IgG, HRP-linked Antibody #7074 is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of Phospho-EGF Receptor (Tyr1068). 
 Antibodies in kit are custom formulations specific to kit.

Background

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 c-Cbl, an adaptor protein that leads 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) provides 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).

1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.
2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.
3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.
4. Hubbard, S.R. et al. (1994) Nature 372, 746-54.
5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.
6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.
9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

注意事项：

Low volume microplate * 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).