Figure 1: Treatment of HeLa cells with IFN-alpha stimulates phosphorylation of Stat3 at Tyr705, detected by PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA kit, #7300, but does not affect the level of total Stat3 protein, detected by a Stat3 antibody via Western analysis. OD450 readings are shown in the top figure, while the corresponding Western blot using Phospho-Stat3 (Tyr705) (3E2) Monoclonal Antibody #9138 or Stat3 antibody, is shown in the bottom figure.
Figure 2: Linear relationship between protein concentration of lysates from IFN-alpha-treated HeLa cells and kit assay optical density readings. HeLa cells (75% confluence) were treated with IFN-alpha (100 ng/ml), and lysed after growth at 37oC for 10 min.
|REACTIVITY||H M R Mk|
|Product Includes||Volume||Solution Color|
|Stat3 Rabbit mAb Coated Microwells||96 tests|
|Phospho-Stat3 (Tyr705) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (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|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-Stat3 (Tyr705) protein. A Stat3 rabbit monoclonal antibody has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-Stat3 proteins are captured by the coated antibody. Following extensive washing, a Phospho-Stat3 (Tyr705) mouse monoclonal antibody is added to detect the captured phospho-Stat3 protein. HRP-linked anti-mouse antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-Stat3 protein.
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-Stat3 (Tyr705) Sandwich ELISA Kit detects endogenous levels of Phospho-Stat3 protein. Using this Sandwich ELISA Kit #7300, a significant induction of phospho-Stat3 can be detected in IFN-α-treated HeLa cells. However, the level of total Stat3 protein remains unchanged, as shown by Western analysis (figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Rat, Monkey
The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).
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