Cell Signaling Technology

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 Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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 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

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.

Sensitivity

Sensitivity

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 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).

  1. Gouilleux, F. et al. (1994) EMBO J. 13, 4361-4369.
  2. Wakao, H. et al. (1994) EMBO J. 13, 2182-2191.
  3. Okutani, Y. et al. (2001) Oncogene 20, 6643-6650.
  4. Demoulin, J.B. et al. (1999) J. Biol. Chem. 274, 25855-25861.
  5. Dentelli, P. et al. (1999) J. Immunol. 163, 2151-2159.
  6. Meinke, A. et al. (1996) Mol. Cell. Biol. 16, 6937-6944.

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