Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit #12001

cell based assay   cell-based assay   elisa   sandwich elisa   screening   smad2   smad3  

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No. Size Price
12001S 1 Kit ( 96 assays ) ¥6,346.00 现货查询 购买询价 防伪查询
Product Includes Volume Solution Color
ELISA Sample Diluent 25 ml Blue
Smad2/3 Mouse mAb Coated Microwells 96 tests
P-Smad2 (Ser465/467)/Smad3 (Ser423/425) Detection Ab 11 ml Green
STOP Solution #7002 11 ml Colorless
TMB Substrate #7004 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
Cell Lysis Buffer (10X) #9803 15 ml Yellowish
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
Sealing Tape 2 sheets

Specificity / Sensitivity

PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit recognizes endogenous levels of phospho-Smad2 (Ser465/467) and Smad3 (Ser423/425) proteins 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.

PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit能够识别内源性水平的人细胞磷酸化Smad2蛋白(丝氨酸(465/467位))和磷酸化Smad3蛋白(丝氨酸(423/425位)),见图1.试剂盒的灵敏性见图2. 本试剂盒用来检测指定物种的蛋白,即通过内部测试确定的指定物种,也可能检测来自其他物种的同源蛋白。


The PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of phospho-Smad2 (Ser465/467) and Smad3 (Ser423/425) 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 Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Detection Antibody is added to detect captured phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) 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 phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) proteins. 
 Antibodies in kit are custom formulations specific to kit.

PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit是固相夹心酶联免疫吸附试验(ELISA),能够识别内源性水平的人细胞磷酸化Smad2蛋白(丝氨酸(465/467位))和磷酸化Smad3蛋白(丝氨酸(423/425位))。用Smad2/3 鼠抗包被微孔板。细胞裂解液孵育后,Smad2/3蛋白(磷酸化和非磷酸化)由包被的抗体捕获。充分洗涤后,加入磷酸化-Smad2 (Ser465/467)/Smad3 (Ser423/425) 检测抗体检测捕获的磷酸化Smad2蛋白(丝氨酸(465/467位))和磷酸化Smad3蛋白(丝氨酸(423/425位))。随后用抗兔IgG, HRP-标记的抗体识别结合的检测蛋白。加入HRP底物TMB进行显色。显色的吸光度值与磷酸化Smad2蛋白(丝氨酸(465/467位))和磷酸化Smad3蛋白(丝氨酸(423/425位))的量成正比。试剂盒中的抗体是针对试剂盒定制的。

ELISA - Western correlation

ELISA - Western correlation

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, but does not affect the level of total Smad2 or Smad3 protein detected by PathScan® Total Smad2/3 Sandwich ELISA Kit #12000. 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进行检测,但用PathScan® Total Smad2/3 Sandwich ELISA Kit #12000检测不影响总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® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit is shown. Starved HeLa cells (85% confluence) were treated with 10 ng/ml of hTGF-β3 #8425 for 30 min at 37ºC.图2. 如图所示,用PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit检测的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。


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).

信号转导分子Smad蛋白家族成员是从细胞表面传输TGF-β信号到细胞核中的一个关键细胞内通路的组成部分。已经定义三种不同类别的Smad蛋白:受体调控型Smads(R-Smads),包括Smad1,2,3,5和8;共同介导型Smad(Co-Smads),Smad4;拮抗或抑制型Smads(I-Smads),Smad6和7(1-5)。激活I型受体与特定的R-Smads联合,并磷酸化一个保守的羧基终端SSXS基序。磷酸化R-Smad从受体解离,并与co-Smad (Smad4)形成异聚体复合物,指导复合物转运至细胞核中。一旦达到细胞核内,Smads可以针对各种DNA结合蛋白,调控转录反应(6-8)。

  1. Heldin, C.H. et al. (1997) Nature 390, 465-71.
  2. Attisano, L. and Wrana, J.L. (1998) Curr Opin Cell Biol 10, 188-94.
  3. Derynck, R. et al. (1998) Cell 95, 737-40.
  4. Massagué, J. (1998) Annu Rev Biochem 67, 753-91.
  5. Whitman, M. (1998) Genes Dev 12, 2445-62.
  6. Wu, G. et al. (2000) Science 287, 92-7.
  7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-7.
  8. Moustakas, A. et al. (2001) J Cell Sci 114, 4359-69.

Application References

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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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