Product Pathways - PathScan ELISA
PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Phospho-Stat5 (Tyr694) Mouse Antibody Coated Microwells | 96 tests | |
| Stat5 Rabbit Detection Antibody | 11 milliliters | Green |
| Anti-Rabbit IgG HRP-Linked Antibody | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | 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 (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
Description
CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat5 when phosphorylated at Tyr694. A Phospho-Stat5 Mouse Antibody* has been coated onto the microwells. After incubation with cell lysates, phospho-Stat5 (Tyr694) is captured by the coated antibody. Following extensive washing, a Stat5 Rabbit Detection Antibody* is added to detect the captured phospho-Stat5 protein. Anti-rabbit IgG, HRP-linked antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of Stat5 phosphorylated at Tyr694.*Antibodies in the kit are custom formulations specific to the kit.
Specificity / Sensitivity
CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113 detects endogenous levels of phospho-Stat5 when phosphorylated at Tyr694. As shown in Figure 1, a significant induction of Stat5 phosphorylation at Tyr694 can be detected in EGF-treated HeLa cells using the Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113. The level of total Stat5 detected by Western analysis remains unchanged.
Sandwich ELISA
Figure 1. Treatment of HeLa cells with EGF stimulates phosphorylation of Stat5 at Tyr694, detected by the PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113, but does not affect the level of total Stat5 protein detected by Western analysis. HeLa cells (80-90% confluent) were starved overnight and treated with 100 ng/mL EGF for 40 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using Stat5 (3H7) Rabbit mAb #9358 (left panel) or Phospho-Stat5 (Tyr694) (14H2) Mouse mAb #9356 (right panel), are shown in the bottom figure.
Sandwich ELISA
Figure 2. The relationship between the protein concentration of the lysate from untreated and EGF-treated HeLa cells and the absorbance at 450 nm is shown.
Background
Stat5 is activated in response to a wide variety of ligands including IL-2, GM-CSF, growth hormone and prolactin. Phosphorylation at Tyr694 is obligatory for Stat5 activation (1,2). This phosphorylation is mediated by Src upon erythropoietin stimulation (3). Stat5 has been found to be constitutively active in some leukemic cell types (4). Phosphorylated Stat5 is found in some endothelial cells treated with IL-3, which suggests its involvement in angiogenesis and cell motility (5). Stat5α and β are independently regulated and activated in various cell types. For instance, both isoforms are activated in response to IFN-α in B cells, but only Stat5α is phosphorylated in response to IFN-α in HeLa cells (6).
- Gouilleux, F. et al. (1994) EMBO J. 13, 4361-4369.
- Wakao, H. et al. (1994) EMBO J. 13, 2182-2191.
- Okutani, Y. et al. (2001) Oncogene 20, 6643-6650.
- Demoulin, J.B. et al. (1999) J. Biol. Chem. 274, 25855-25861.
- Dentelli, P. et al. (1999) J. Immunol. 163, 2151-2159.
- Meinke, A. et al. (1996) Mol. Cell. Biol. 16, 6937-6944.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 9351 Phospho-Stat5 (Tyr694) Antibody
- 9359 Phospho-Stat5 (Tyr694) (C11C5) Rabbit mAb
- 9356 Phospho-Stat5 (Tyr694) (14H2) Mouse mAb
- 9358 Stat5 (3H7) Rabbit mAb
- 9363 Stat5 Antibody
- 9310 Stat5 Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
