Cell Signaling Technology

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).

Species Cross-Reactivity

H M Mi

Reactivity Key:  H=Human  M=Mouse  Mi=Mink
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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

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.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of lysates from untreated and TGF-β-treated HaCaT cells and the absorbance at 450 nm is shown. Cells (85% confluence) were treated with 10 ng/ml TGF-β for 30 min. at 37oC.

Background

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).

  1. Heldin, C.H. et al. (1997) Nature 390, 465-471.
  2. Attisano, L. and Wrana, J.L. (1998) Curr. Opin. Cell Biol. 10, 188-194.
  3. Derynck, R. et al. (1998) Cell 95, 737-740.
  4. Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791.
  5. Whitman, M. (1998) Genes Dev. 12, 2445-2462.
  6. Wu, G. et al. (2000) Science 287, 92-97.
  7. Attisano, L. and Wrana, J.L. (2002) Science 296, 1646-1647.
  8. Moustakas, A. et al. (2001) J. Cell Sci. 114, 4359-4369.

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