PathScan? Phospho-PDGF Receptor β (Tyr751) Sandwich ELISA Kit

• 产品编号：CST-7345C      品牌：CST       原厂货号：7345C
• 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-PDGF Receptor beta (Tyr751) Sandwich ELISA Kit主要用于检测内源磷酸化PDGF Receptor beta (Tyr751)的表达。使用 夹心法ELISA试剂盒#7345能够检测到PDGF刺激过的NIH/3T3 细胞中磷酸化PDGF Receptor beta (Tyr751)表达的显著升高。而用PDGF Receptor beta 兔单抗 (7345-28D12)通过免疫印记 法对总PDGF Receptor beta 的检测结果没有改变(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。CST公司的PathScan® Phospho-PDGF Receptor beta (Tyr751) Sandwich ELISA Kit 利用固相双抗夹心酶联免疫检测法对内源性磷酸化PDGF Receptor beta (Tyr751) 蛋白的表达进行检测。微孔中已经包被了PDGF Receptor beta (7345-28D12) 兔单抗*，与细胞裂解液共孵育后，磷酸化的和未磷酸化的PDGF Receptor beta均能被包被抗体捕获。多次洗板之后，加入磷酸化PDGF Receptor beta鼠单抗#3166**对被捕获的磷酸化的和未磷酸化的磷酸化PDGF Receptor beta蛋白进行检测，然后加入抗鼠IgG, HRP偶联抗体 #7076*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，发射光强度与磷酸化PDGF Receptor beta (Tyr751) 蛋白的量成正比。试剂盒中的抗体为试剂盒特别订制配方。血小板衍生生长因子(PDGF) 家族的蛋白以几种二硫键连接的二聚体形式存在 (PDGF AA, PDGF AB, PDGF BB, PDGF CC和PDGF DD)，它们以特定的方式与两种密切相关的酪氨酸激酶受体-PDGF receptor α (PDGFRα)和PDGF receptor β (PDGFRβ)-结合。PDGFRα 和PDGFRβ 的胞内激酶区有75% 到85% 的序列同源性，而激酶插入区和羧基端仅有很低的同源性(27% 到 28%) (1). PDGFRα 同型二聚体和除了含有PDGF D 之外的所有的PDGF亚型结合。PDGFRβ同型二聚体能结合PDGF BB 和DD亚型，还有PDGF AB 异二聚体。 异聚体 PDGF receptor α/β能结合 PDGF B, C, 和 D 同型二聚体，还有 PDGF AB 异二聚体 (2)。 PDGFRα 和 PDGFRβ 都能被PDGF激活和 EGFR形成异二聚体, (3)。各种细胞的受体总数、受体亚基组成都不相同，因此不同细胞对PDGF的反应也不同(4)。配体结合能诱发受体的二聚化和自磷酸化，继而结合并激活胞内含有SH2结构的信号传导分子， 如Grb2, Src, GAP, PI3K激酶, PLCγ和 Nck。激活的PDGFRs能引发一系列不同的信号通路 并影响细胞的生长、肌动蛋白重塑、迁移和分化 (5)。PDGFRβ激酶插入区的Tyr751位点结合PI3K激酶 (6)。根据PDGFRβ的Tyr751位点附近序列产生的磷酸化五肽(pTyr751-Val-Pro-Met-Leu)能够抑制PI3K激酶p85亚基的羧基端SH2区和PDGFRβ结合 (7)。 Tyr740对 PDGFRβ介导的 PI3 K激酶的激活也是必需的(8)。

1. Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
2. Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
3. Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
4. Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
5. Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
6. Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
7. Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.

原厂资料：

Specificity / Sensitivity

CST's PathScan® Phospho-PDGF Receptor beta (Tyr751) Sandwich ELISA Kit detects endogenous levels of Phospho-PDGF Receptor beta (Tyr751) protein. Using this Sandwich ELISA Kit #7345, a significant induction of phospho-PDGF Receptor beta (Tyr751) in NIH/3T3 cells treated with PDGF can be detected. However, the level of total PDGF Receptor beta protein remains unchanged, as shown by Western analysis using PDGF Receptor beta Rabbit mAb (7345-28D12) [Fig.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

CST's PathScan® Phospho-PDGF Receptor beta (Tyr751) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-PDGF Receptor beta (Tyr751) protein. A PDGF Receptor beta (7345-28D12) Rabbit mAb* has been coated onto the microwells. After incubation with cell lysates, Both nonphospho- and phospho- PDGF Receptor beta proteins are captured by the coated antibody. Following extensive washing, Phospho-PDGF Receptor beta Mouse mAb #3166* is added to detect the captured phospho-PDGF Receptor beta protein. HRP-linked anti-mouse antibody #7076* 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 phospho-PDGF Receptor beta (Tyr751) protein. 
 * Antibodies in the kit are custom formulations specific to the kit

Background

The proteins of the platelet derived growth factor (PDGF) family exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC, and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β binds PDGF B, C, and D homodimers as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules such as Grb2, Src, GAP, PI3 kinase, PLCγ, and Nck. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration, and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ-mediated PI3 kinase activation (8).

1. Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
2. Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
3. Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
4. Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
5. Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
6. Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
7. Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.

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).