Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Stat1 (Tyr701) Sandwich ELISA Kit #7234

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Kit Includes Volume Solution Color
Stat1 Ab Coated Microwells 96 tests
P-Stat1 (Y701) Detection Ab 11 ml Green
HRP-linked Streptavidin 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

The PathScan® Phospho-Stat1 (Tyr701) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat1 when phosphorylated at Tyr701. A Stat1 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, Stat1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, biotinylated Phospho-Stat1 (Tyr701) Rabbit Detection Antibody is added to detect phosphorylation of Tyr701 on the captured Stat1 protein. HRP-linked streptavidin 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 Stat1 phosphorylated at Tyr701.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-Stat1 (Tyr701) Sandwich ELISA Kit detects endogenous levels of Phospho-Stat1 when phosphorylated at Tyr701. As shown in Figure 1, a significant induction of Stat1 phosphorylation at Tyr701 can be detected in HeLa cells following treatment with Interferon-γ (IFN-γ) using the Phospho-Stat1 (Tyr701) Sandwich ELISA Kit #7234. The level of total Stat1 (phospho and nonphospho) remains unchanged as shown by Western analysis. 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 IFN-γ stimulates phosphorylation of Stat1 at Tyr701, detected by the PathScan® Phospho-Stat1 (Tyr701) Sandwich ELISA Kit #7234, but does not affect the level of total Stat1 protein detected by Western analysis. HeLa cells (80-90% confluent) were starved for 6 hours and treated with 100 ng/mL IFN-γ for 20 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using Stat1 (42H3) Rabbit mAb (Human Specific) #9175 (left panel) or Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 (right panel), are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between lysate protein concentration from untreated and IFN-γ -treated HeLa cells and the absorbance at 450 nm is shown.

Background

The Stat1 transcription factor is activated in response to a large number of ligands (1) and is essential for responsiveness to IFN-α and IFN-γ (2,3). Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation, and DNA binding (4). Stat1 protein exists as a pair of isoforms, Stat1α (91 kDa) and the splice variant Stat1β (84 kDa). In most cells, both isoforms are activated by IFN-α, but only Stat1α is activated by IFN-γ. The inappropriate activation of Stat1 occurs in many tumors (5). In addition to tyrosine phosphorylation, Stat1 is also phosphorylated at Ser727 through a p38 mitogen-activated protein kinase (MAPK)-dependent pathway in response to IFN-α and other cellular stresses (6). Serine phosphorylation may be required for the maximal induction of Stat1-mediated gene activation.

  1. Heim, M.H. (1999) J. Recept. Signal. Transduct. Res. 19, 75-120.
  2. Durbin, J.E. et al. (1996) Cell 84, 443-450.
  3. Meraz, M.A. et al. (1996) Cell 84, 431-442.
  4. Ihle, J.N. et al. (1994) Trends Biochem. Sci. 19, 222-227.
  5. Frank, D.A. (1999) Mol. Med. 5, 432-456.
  6. Wen, Z. et al. (1995) Cell 82, 241-250.

Application References

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Protocols

Companion Products

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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