| 货号 | 7355C |
| 描述 | CSTs PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-IκBα (Ser32) protein. An IκBα Mouse mAb #7361* has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-IκBα proteins are captured by the coated antibody. Following extensive washing, Phospho-IκBα (Ser32) Antibody #2859* is added to detect the captured phospho-IκBα (Ser32) protein. HRP-linked anti-rabbit antibody #7074* 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-IκBα (Ser32) protein. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit detects endogenous levels of Phospho-IκBα (Ser32) protein. As shown in Figure 1, using this Sandwich ELISA Kit #7355, a significant induction of Phospho-IκBα (Ser32) in HeLa cells treated with TNF-α can be detected. However, the level of total IκBα (phospho- and nonphospho-), detected by the Total IκBα Sandwich ELISA Kit #7360, remains unchanged. 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 NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins (1-3). Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified (8). |
| 存放说明 | 4C |
| 参考文献 | Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70. Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8. Brown, K. et al. (1995) Science 267, 1485-8. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83. Chen, Z.J. et al. (1996) Cell 84, 853-62. Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63. |
Figure 1: Treatment of HeLa cells with TNF-α stimulates phosphorylation of IκBα at Ser32, detected by PathScan® Phospho-IκBα (Ser32) Sandwich ELISA kit, #7355, but does not affect the level of total IκBα protein detected by PathScan® Total IκBα Sandwich ELISA kit, #7360. Treatment with MG132, a proteasome inhibitor (37ºC for 180 min before TNF-α induction), causes accumulation of phospho-IκBα in control and TNF-α-treated cells, shown in both Sandwich ELISA and Western blot analysis. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-IκBα (Ser32) Ab #9241 (right panel) or IκBα (L27H11) Mouse mAb #7361 (left panel), is shown in the bottom figure.图1: PathScan® Phospho-IκBα (Ser32) Sandwich ELISA kit, #7355检测经TNF-α诱导的HeLa细胞中在Ser32位点磷酸化的IκBα ，但是经PathScan® Total IκBα Sandwich ELISA kit, #7360检测证明处理并不影响总IκBα蛋白。在TNF-α诱导处理前，用蛋白酶抑制剂MG132在37ºC 条件下处理180min , 引起对照和 TNF-α处理的细胞中磷酸化的IκBα的积累，这均通过Sandwich ELISA 和 Western免疫印迹分析。OD450的读数在图片的最上面显示,同时对应的Western免疫印迹在下面图片显示，所用抗体为Phospho-IκBα (Ser32) Ab #9241(右侧图)或IκBα (L27H11) Mouse mAb #7361(左侧图)。 | |
Figure 2: Linear relationship between protein concentration of lysates from untreated and TNF-α-treated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluence) were treated with TNF-α (10 ng/ml) and lysed after incubation at 37ºC for 5 minutes.图2.未经处理和经TNF-α处理的HeLa细胞的裂解液蛋白浓度与试剂盒实验最佳光密度读数之间的关系。HeLa 细胞(70-85% confluence) 在37ºC 条件下经TNF-α (10 ng/ml)孵育5min，并裂解。 |
