Product Pathways - PathScan ELISA
PathScan® Phospho-Smad3 (Ser423/425) Sandwich ELISA Kit #12003
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|Kit Includes||Volume||Solution Color|
|Smad2/3 Antibody Coated Microwells||96 tests|
|P-Smad3 (Ser423/425) Detection Ab||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
H M Mi
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The PathScan® Phospho-Smad3 (Ser423/425) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of Smad3 (Ser423/425) protein. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Phospho-Smad3 (Ser423/425) Rabbit Detection Antibody is added to detect captured 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-Smad3 (Ser423/425) proteins.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Phospho-Smad3 (Ser423/425) Sandwich ELISA Kit recognizes endogenous levels of phospho-Smad3 (Ser423/425) 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.
ELISA - Western correlation
Figure 1. Treatment of HeLa cells with hTGF-β3 #8425 stimulates phosphorylation of Smad3 at Ser423/425, as detected by PathScan® Phospho-Smad3 (Ser423/425) Sandwich ELISA Kit, but does not affect the level of total Smad3 protein detected by PathScan® Total Smad3 Sandwich ELISA Kit #12002. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Smad3 (C67H9) Rabbit mAb #9523 (left panel) and a phospho-Smad3 (Ser423/425) rabbit mAb (right panel) are shown in the bottom figure.
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-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.
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).
- Heldin, C.H. et al. (1997) Nature 390, 465-471.
- Attisano, L. and Wrana, J.L. (1998) Curr. Opin. Cell Biol. 10, 188-194.
- Derynck, R. et al. (1998) Cell 95, 737-740.
- Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791.
- Whitman, M. (1998) Genes Dev. 12, 2445-2462.
- Wu, G. et al. (2000) Science 287, 92-97.
- Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-1647.
- Moustakas, A. et al. (2001) J. Cell Sci. 114, 4359-4369.
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- 12000 PathScan® Total Smad2/3 Sandwich ELISA Kit
- 12001 PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit
- 12002 PathScan® Total Smad3 Sandwich ELISA Kit
- 7348 PathScan® Phospho-Smad2 (Ser465/467) Sandwich ELISA Kit
- 7244 PathScan® Total Smad2 Sandwich ELISA Kit
- 9513 Smad3 Antibody
- 9523 Smad3 (C67H9) Rabbit mAb
- 9520 Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 8425 Human Transforming Growth Factor β3 (hTGF-β3)
- 7004 TMB Substrate
- 7002 STOP Solution
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9998 BSA
For Research Use Only. Not For Use In Diagnostic Procedures.