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.
Figure 2. The relationship between lysate protein concentration from untreated and IFN-γ -treated HeLa cells and the absorbance at 450 nm is shown.
YES! Please alert me when the status of this product changes.
|Product Includes||Volume||Solution Color|
|Stat1 Rabbit mAb Coated Microwells||96 tests|
|Phospho-Stat1 (Tyr701) Rabbit Detection mAb (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
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.
Explore pathways + proteins related to this product.
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.