Product Pathways - PathScan ELISA
PathScan® Phospho-Smad2 (Ser465/467) Sandwich ELISA Kit #7348
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|Kit Includes||Volume||Solution Color|
|Smad2 Mouse mAb Coated Microwells||96 tests|
|Phospho-Smad2 (Ser465/467) Detection Rabbit mAb||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml|
|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-Smad2 (Ser465/467) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Smad2 when phosphorylated at Ser465/467. A Smad2 Mouse monoclonal Antibody has been coated onto the microwells. After incubation with cell lysates, Smad2 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Smad2 (Ser465/467) Detection Antibody is added to detect serine phosphorylation of the captured Smad2 protein. Anti-rabbit IgG, HRP-Linked Ab 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 phosphorylated at Ser465/467.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-Smad2 (Ser465/467) Sandwich ELISA Kit #7348 detects Smad2 when phosphorylated at Ser465/467. As shown in Figure 1, a significant induction of Smad2 phosphorylation at Ser465/467 can be detected in TGF-β-treated HaCaT cells using the PathScan® Phospho-Smad2 (Ser465/467) Sandwich ELISA Kit #7348. The level of total Smad2 (phospho and nonphospho) remains unchanged as shown by western analysis and by PathScan® Total Smad2 Sandwich ELISA Kit #7244 (Figure 1). 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 HaCaT cells with TGF-β stimulates phosphorylation of Smad2 at Ser465/467, detected by PathScan® Phospho-Smad2 (Ser465/467) Sandwich ELISA Kit #7348, but does not affect the level of total Smad2 protein detected by PathScan® Total Smad2 Sandwich ELISA Kit #7244. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Smad2 (86F7) Rabbit mAb #3122 (left panel) or Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (right panel) are shown in the bottom figure.
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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- 7244 PathScan® Total Smad2 Sandwich ELISA Kit
- 3104 Phospho-Smad2 (Ser245/250/255) Antibody
- 3108 Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb
- 3101 Phospho-Smad2 (Ser465/467) Antibody
- 5339 Smad2 (D43B4) XP® Rabbit mAb
- 3122 Smad2 (86F7) Rabbit mAb
- 3103 Smad2 (L16D3) Mouse mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.