Revision 21

#12001

Store at +4C

PathScan® Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit

1 x 96 tests

Species Cross Reactivity:
H M Mi

UniProt ID:
#P84022, #Q15796

Entrez-Gene Id:
#4088, #4087

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Color Storage Temp
SMAD2/3 Mouse Monoclonal Antibody Coated Microwells, 384 Well ("D" Kit Only)19503384 tests+4C
SMAD2/3 Mouse Monoclonal Antibody Coated Microwells6168496 tests+4C
Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Rabbit Detection Antibody339251 eaGreen (Lyophilized)+4C
Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)132721 eaRed (Lyophilized)+4C
Detection Antibody Diluent1333911 mlGreen+4C
HRP Diluent1351511 mlRed+4C
TMB Substrate700411 ml+4C
STOP Solution700211 ml+4C
Sealing Tape545032 ea+4C
ELISA Wash Buffer (20X)980125 ml+4C
ELISA Sample Diluent1108325 mlBlue+4C
Cell Lysis Buffer (10X)980315 ml-20C

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

For the “C” and “V” kits, the supplied 96-well strip plate consists of twelve 8-well strips in a support frame. This enables custom plate configurations.

For the “D” kits, the supplied 384-well microplate is a single piece of material and does not break apart.

Description

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 this kit are custom formulations specific to kit.

Specificity/Sensitivity

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. This 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.

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 9; 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-SMADs dissociate from the receptor and form a heteromeric complex with SMAD4, initiating translocation of the heteromeric SMAD complex to the nucleus. Once in the nucleus, SMADs recruit a variety of DNA binding proteins that function to regulate transcriptional activity (6-8).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

使用に関する制限

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Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 21

Cell Signaling Technology Logo
Figure 1. Treatment of HeLa cells with hTGF-β3 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.
ELISA and/or ELISA-like Assays Image 1: PathScan<sup>®</sup> Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit
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 for 30 min at 37ºC.
ELISA and/or ELISA-like Assays Image 2: PathScan<sup>®</sup> Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit
Figure 3. The relationship between the protein concentration of the lysate from untreated and TGF-β3-treated HT1080 cells and the absorbance at 450 nm is shown for the 384-well kit format. Starved HeLa cells (85% confluence) were treated with 10 ng/ml of hTGF-β3 for 30 min at 37ºC.
ELISA and/or ELISA-like Assays Image 3: PathScan<sup>®</sup> Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.