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.
|REACTIVITY||H M Mi|
|Product Includes||Volume||Solution Color|
|Smad2/3 Mouse mAb Coated Microwells||96 tests|
|Phospho-Smad3 (Ser423/425) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
Human, Mouse, Mink
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).
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