Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Smad2 Sandwich ELISA Kit #7244

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Kit Includes Volume Solution Color
Smad2 mAb coated microwells 96 tests
Smad2 Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Sealing Tape 2 sheets
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 Mi

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

Description

CST's PathScan® Total Smad2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Smad2 protein. A Smad2 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Smad2 proteins are captured by the coated antibody. Following extensive washing, Smad2 Mouse mAb is added to detect both the captured phospho- and nonphospho-Smad2 protein. Anti-mouse IgG, HRP-linked Antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Smad2 protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total Smad2 Sandwich ELISA Kit #7244 detects endogenous levels of total Smad2 protein. As shown in Figure 1, both phospho- and nonphospho-Smad2 proteins from untreated and TGFβ treated MV1LU cell lysates are detected by this kit. In Figure 3, Western blot analysis of protein captured in the Smad2 Rabbit mAb #3109 coated microwell shows a single band corresponding to the Smad2 protein. Smad2 in Hela cells also can be detected by this kit (data not shown). 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: Nonphospho and phospho-Smad2 proteins from untreated and TGF-β treated Mv1Lu cells detected by PathScan® Total Smad2 Sandwich ELISA kit #7244 showing similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blots using Smad2 Mouse mAb #3103 (left panel) or Phospho-Smad2 (Ser465/467) Antibody #3101 (right panel), are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and TGF-β-treated Mv1Lu cells and kit assay optical density readings. After starvation, Mv1Lu cells (85% confluence) were treated with TGF-β (100 ng/ml) for 15-30 min at 37°C, and then lysed.

Specificity

Specificity

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA well captured protein is shown. Lysates were prepared from mink Mv1Lu cells and incubated in wells coated with capture antibody #3109. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. Mv1Lu lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using Smad2 Mouse mAb #3119. A single band corresponding to the Smad2 protein is detected in the captured material (lane 2).


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.

Application References

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Protocols

Companion Products

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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