Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Smad2/3 Sandwich ELISA Kit #12000

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

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No. Size Price
12000C 1 Kit ( 96 assays ) ¥6,345.00 现货查询 购买询价
Product Includes Volume Solution Color
ELISA Sample Diluent 25 ml Blue
Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated) 1 ea Red (Lyophilized)
Detection Antibody Diluent 11 ml Green
HRP Diluent 11 ml Red
Smad2/ 3 Rabbit Detection mAb 1 ea Green (Lyophilized)
Smad2/3 Antibody Coated Microwells 96 tests
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
Sealing Tape 2 sheets

Specificity / Sensitivity

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.

PathScan® Total Smad2/3 Sandwich ELISA Kit 能够识别内源性水平的人细胞Smad2和Smad3蛋白,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蛋白,也可能检测来自其他物种的同源蛋白。


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.

PathScan® Total Smad2/3 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Smad2和Smad3蛋白。Smad2/3鼠抗包被微孔板。细胞裂解液孵育后,Smad2/3蛋白(磷酸和非磷酸)由包被的抗体捕获。充分冲洗后,加入Smad2/3 兔检测抗体检测捕获的Smad2/3蛋白。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Smad2和Smad3蛋白的量成正比。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 #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。


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。已经确定了三种不同类别的Smad蛋白:受体调控型Smads(R-Smads),其中包括Smad1,2,3,5和8;共同介质型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,允许复合物转移至细胞核中。一旦达到细胞核内,Smads可以靶向各种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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