Figure 1. Treatment of HeLa cells with hTGF-β3 #8425 stimulates phosphorylation of Smad2 at Ser465/467 or Smad3 at Ser423/425, as detected by PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit, but does not affect the level of total Smad2 or Smad3 protein detected by PathScan® Total Smad2/3 Sandwich ELISA Kit #12000. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Smad2/3 (D7G7) XP® Rabbit mAb #8685 (left panel), Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (center panel), or 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-Smad2 (Ser465/467)/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-Smad2 (Ser465/467)/Smad3 (Ser423/425) Rabbit Detection Antibody||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-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of phospho-Smad2 (Ser465/467) and Smad3 (Ser423/425) proteins. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad2/3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Detection Antibody is added to detect captured phospho-Smad2 (Ser465/467) and 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-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) proteins.
Antibodies in kit are custom formulations specific to kit.
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
Protocol Id: 21
PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit recognizes endogenous levels of phospho-Smad2 (Ser465/467) and Smad3 (Ser423/425) proteins 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).
Explore pathways + proteins related to this product.