PathScan? Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit

• 产品编号：CST-7315C      品牌：CST       原厂货号：7315C
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
• 应用分类：

其它组分：

描述：

CST的PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315能够检测Thr202/Tyr204磷酸化的phospho-p44 MAP Kinase蛋白水平。如图1所示，使用Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315能检测到经PDGF处理的NIH/3T3细胞中p44 MAPK的Thr202/Tyr204磷酸化被显著诱导。用western分析发现p44 MAP Kinase (Erk1)总蛋白水平没有发生改变。注意：如果加入到微孔中的稀释后裂解物中含有终浓度为0.5%的脱氧胆酸钠或0.05% SDS，吸光度的读值将会增加。该试剂盒经公司内部实验证明能检测已标明物种的蛋白，但也可能能检测其他物种中的类似蛋白。CST的 PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit是通过固相夹心酶联免疫吸附法（ELISA）来检测内源性的Thr202/Tyr204位点磷酸化的p44 MAPK(Erk1)蛋白水平。先将 p44 MAP Kinase Rabbit Antibody包被在微孔板上。与细胞裂解物孵育后，p44 MAPK蛋白(磷酸化及非磷酸化）便可被包被的抗体捕获。清洗彻底后，加入phospho-p44/42 MAPK (Thr202/Tyr204) Mouse Detection Antibody检测Thr202/Tyr204位点磷酸化的p44 MAPK(Erk1)蛋白水平。再加入Anti-Rabbit IgG, HRP偶联抗体#7076作为二抗来识别之前绑定的检测抗体。最后加入HRP底物—— TMB，进行显色。产生的吸光值与Phospho-p44 MAPK (Thr202/Tyr204)蛋白的数量成正比。*试剂盒中抗体是专为本试剂盒特制的。丝裂原活化蛋白激酶(MAPKs)是一个具有广泛保守性的丝氨酸/苏氨酸蛋白激酶家族，在许多细胞过程中起作用，比如细胞增殖、分化、运动和死亡。p44/42 MAPK (Erk1/2)信号转导通路能在应答各种胞外刺激后被激活，这些刺激因子包括有丝分裂原、生长因子和细胞因子(1-3)，并且被研究者认为是诊断和治疗癌症的重要靶点(4)。受到刺激后，三个不同层次的蛋白激酶级联反应就连续被开启，这三个层次的分子包含MAPKKK（MAP3K）、MAPKK（MAP2K）、MAPK。多种p44/42 MAP3Ks已经被鉴定出来，包括Raf家族成员，以及Mos、Tpl2/Cot。MEK1、MEK2是该通路中主要的MAPKKs(5,6)。MEK1、MEK2通过分别磷酸化p44和p42蛋白的活化环内的Thr202/Tyr204位点和Thr185位点/Tyr187位点，从而激活p44、p42。目前已经鉴定出p44/42的一些下游靶点，包括p90RSK (7) 和下游转录因子Elk-1 (8,9)。双特异性(Thr/Tyr) MAPK磷酸酶家族成员DUSPs、MKPs (10)，以及MEK抑制子如U0126、 PD98059，可以负调节p44/42。

1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
8. Marais, R. et al. (1993) Cell 73, 381-93.
9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
11. Emre, Y. et al. (2007) Biochem J 402, 271-8.
12. Cuthbert, P.C. et al. (2007) J Neurosci 27, 2673-82.

原厂资料：

Specificity / Sensitivity

CST’s PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315 detects endogenous levels of phospho-p44 MAP Kinase when phosphorylated at Thr202/Tyr204. As shown in Figure 1, a significant induction of p44 MAP Kinase (Erk1) phosphorylation at Thr202/Tyr204 can be detected in PDGF-treated NIH/3T3 cells using the Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315. The level of total p44 MAP Kinase (Erk1) detected by Western analysis remains unchanged. Note: This assay may have an increased absorbance reading if the diluted lysate that is applied to the microwells contains a final concentration of 0.5% sodium deoxycholate or 0.05% SDS. 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-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p44 MAP Kinase (Erk1) when phosphorylated at Thr202/Tyr204. A p44 MAP Kinase Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, p44 MAP Kinase (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-p44/42 MAPK (Thr202/Tyr204) Mouse Detection Antibody is added to detect phosphorylation of Thr202/Tyr204 on the captured p44 MAP Kinase (Erk1) protein. Anti-mouse IgG, HRP-linked antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of p44 MAP Kinase (Erk1) phosphorylated at Thr202/Tyr204. 
 Antibodies in the kit are custom formulations specific to the kit.

Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3) and is an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family as well as Mos and Tpl2/Cot. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors such as U0126 and PD98059.

1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
8. Marais, R. et al. (1993) Cell 73, 381-93.
9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
11. Emre, Y. et al. (2007) Biochem J 402, 271-8.
12. Cuthbert, P.C. et al. (2007) J Neurosci 27, 2673-82.

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).