| 货号 | 12000C-CST |
| 描述 | The PathScan® Total Smad2/3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of Smad2 and Smad3 proteins. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad2/3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Smad2/3 Rabbit Detection Antibody is added to detect captured Smad2/3 proteins. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Smad2 and Smad3 proteins. Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Mouse/Mink |
| 应用 | ELISA |
| 目标/特异性 | PathScan® Total Smad2/3 Sandwich ELISA Kit recognizes endogenous levels of total Smad2 and Smad3 protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. 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 | Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8). |
| 存放说明 | 4C |
| 参考文献 | Heldin, C.H. et al. (1997) Nature 390, 465-71. Attisano, L. and Wrana, J.L. (1998) Curr Opin Cell Biol 10, 188-94. Derynck, R. et al. (1998) Cell 95, 737-40. Massagué, J. (1998) Annu Rev Biochem 67, 753-91. Whitman, M. (1998) Genes Dev 12, 2445-62. Wu, G. et al. (2000) Science 287, 92-7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-7. Moustakas, A. et al. (2001) J Cell Sci 114, 4359-69. |
Figure 1. Treatment of HeLa cells with hTGF-β3 #8425 stimulates phosphorylation of Smad2 at Ser465/467 or Smad3 at Ser423/425, as detected by PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit #12001, but does not affect the level of total Smad2 or Smad3 protein detected by PathScan® Total Smad2/3 Sandwich ELISA Kit. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Smad2/3 (D7G7) XP® Rabbit mAb #8685 (left panel), Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (center panel), or a phospho-Smad3 (Ser423/425) Rabbit mAb (right panel) are shown in the bottom figure. 图1. HeLa细胞用hTGF-β3 #8425处理，刺激Smad2丝氨酸（465/467位）或Smad3丝氨酸（423/425位）磷酸化，用PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit #12001进行检测，但不影响用PathScan® Total Smad2/3 Sandwich ELISA Kit检测总Smad2 或Smad3蛋白水平。上图显示在450nm处的吸光度值，而相应的western blots结果见下图，western blots实验所用的抗体为Smad2/3 (D7G7) XP® Rabbit mAb #8685 (左面板), Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (中面板)或 phospho-Smad3 (Ser423/425) Rabbit mAb (右面板)。 | |
Figure 2. The relationship between the protein concentration of lysates from untreated and TGF-β3-treated HeLa cells and the absorbance at 450 nm as detected by the PathScan® Total Smad2/3 Sandwich ELISA Kit is shown. Starved HeLa cells (85% confluence) were treated with 10 ng/ml hTGF-β3 #8425 for 30 min at 37ºC. 图2. 如图所示，用PathScan® Total Smad2/3 Sandwich ELISA Kit 检测HeLa细胞未经处理或用TGF-β3处理后裂解物蛋白浓度与450 nm处吸光度值的关系。饥饿处理的HeLa细胞（85%汇合）用10 ng/ml hTGF-β3 #8425 37ºC处理30min。 |
