| 货号 | 7175C |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA Kit detects endogenous levels of phospho-MEK1 protein. A significant induction of MEK1 phosphorylation in PMA-treated NIH/3T3 cells can be detected using this kit, #7175. However, the level of total MEK1 protein detected by PathScan® Total MEK1 Sandwich ELISA Kit #7165 remains unchanged (Figure 1). This kit can also be used to detect phosphorylated MEK1 protein in human 293 cells. 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 | MEK1 and MEK2, also called MAPK or Erk kinases, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation (1-3). Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221, located in the activation loop of subdomain VIII, by Raf-like molecules. MEK1/2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx (1-4). Constitutively active forms of MEK1/2 are sufficient for the transformation of NIH/3T3 cells or the differentiation of PC-12 cells (4). MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII. |
| 存放说明 | 4C |
| 参考文献 | Crews, C.M. et al. (1992) Science 258, 478-480. Alessi, D.R. et al. (1994) EMBO J. 13, 1610-19. Rosen, L.B. et al. (1994) Neuron 12, 1207-21. Cowley, S. et al. (1994) Cell 77, 841-52. |
Figure 1: Treatment of NIH/3T3 cells with PMA stimulates phosphorylation of MEK1 at Ser217/221, detected by PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA kit #7175, but does not affect the level of total MEK1 protein detected by PathScan® Total MEK1 Sandwich ELISA kit #7165. Lambda protein phosphatase (LPP) treatment of control cell lysates (37ºC for 90 minutes) abolished the basal phosphorylation of MEK1 in control lysates shown in both Sandwich ELISA and Western analysis. The OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-MEK1/2 (Ser217/221) Antibody #9121 (right panel) or MEK1 Mouse mAb #2352 (left panel), is shown in the bottom figure.图1：用PMA刺激NIH/3T3细胞后使MEK1蛋白丝氨酸（217/221位点）磷酸化，检测试剂盒是PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA kit #7175，但是总MEK1蛋白水平并没有受到影响，检测试剂盒是 PathScan® Total MEK1 Sandwich ELISA kit #7165。Sandwich ELISA 和Western检测分析都显示用Lambda蛋白磷酸酶（LPP）处理对照细胞裂解物（37ºC，90 min）后，消除了对照裂解物中MEK1蛋白的基础磷酸化。上图显示OD450读数，下图是对应的Western blot检测结果，使用的抗体是 Phospho-MEK1/2 (Ser217/221) Antibody #9121 (右侧) 和MEK1 Mouse mAb #2352(左侧)。 | |
Figure 2: Linear relationship between protein concentration of lysates from untreated and PMA-treated NIH/3T3 cells and kit assay optical density readings. Cells (80% confluence) were treated with PMA (120 ng/ml) and lysed after incubation at 37ºC for 30 minutes. Lambda protein phosphatase (LPP) treatment of control cell lysates was performed at 37ºC for 90 minutes.图2：未处理、PMA处理的293细胞裂解物中蛋白质浓度和试剂盒分析光学密度读数之间的线性关系。PMA (120 ng/ml)处理的细胞（80%生长汇合）37ºC孵育30min后再裂解。Lambda蛋白磷酸酶（LPP）37ºC 90 min处理对照细胞裂解物。 |
