Product Pathways - PathScan ELISA
PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113
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|Kit Includes||Volume||Solution Color|
|P-Stat5 (Y694) Ab Coated Microwells||96 tests|
|Stat5 Detection Ab||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|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 enclosed in parentheses are predicted to react based on 100% sequence homology.
CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat5a when phosphorylated at Tyr694 and Stat5b at Tyr699. A Phospho-Stat5 Mouse Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Stat5a (Tyr694) and phospho-Stat5b (Tyr699) proteins are captured by the coated antibody. Following extensive washing, a Stat5 Rabbit Detection Antibody is added to detect the captured phospho-Stat5a and phospho-Stat5b 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 Stat5a phosphorylated at Tyr694 and Stat5b phosphorylated at Tyr699.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113 detects endogenous levels of phospho-Stat5a when phosphorylated at Tyr694 and Stat5b at Tyr699. As shown in Figure 1, a significant induction of Stat5 phosphorylation 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. 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
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.
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 is 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). Stat5a and Stat5b are independently regulated and activated in various cell types. For instance, interferon treatment predominantly activates Stat5a in U-937 cells and Stat5b 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.
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- 4322 Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb
- 9359 Phospho-Stat5 (Tyr694) (C11C5) Rabbit mAb
- 9356 Phospho-Stat5 (Tyr694) (14H2) Mouse mAb
- 9351 Phospho-Stat5 (Tyr694) Antibody
- 9358 Stat5 (3H7) Rabbit mAb
- 9363 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)
For Research Use Only. Not For Use In Diagnostic Procedures.