Product Pathways - PathScan ELISA

PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit #7300

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Kit Includes	Volume	Solution Color
Stat3 (7300-52F7) Rabbit Monoclonal Ab Coated Microwells
Phospho-Stat3 (Tyr705) (3E2) Mouse Monoclonal Detection Ab	11 milliliters	Green
Anti-mouse IgG HRP-Linked Ab	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
Sealing Tape	2 sheets
20X Wash Buffer	25 milliliters	Colorless
Sample Diluent	25 milliliters	Blue
Cell Lysis Buffer (10X) # 9803	15 milliliters	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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human

Description

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 (7300-52F7*) 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 mouse monoclonal antibody #9138* is added to detect the captured phospho-Stat3 protein. HRP-linked anti-mouse antibody #7076* 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 the kit are custom formulations specific to the kit.

Specificity / Sensitivity

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-alpha-treated HeLa cells. However, the level of total Stat3 protein remains unchanged, as shown by Western analysis using Stat3 rabbit monoclonal antibody (7300-52F7) (figure 1).

Background

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). 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).

1. Heim, M.H. (1999) J. Recept. Signal Transduct. Res. 19, 75-120.
2. Takeda, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3801-3804.
3. Catlett-Falcone, R. et al. (1999) Immunity 10, 105-115.
4. Garcia, R. and Jove, R. (1998) J. Biomed. Sci. 5, 79-85.
5. Bromberg, J.F. et al. (1999) Cell 98, 295-303.
6. Darnell Jr., J.E. et al. (1994) Science 264, 1415-1421.
7. Ihle, J.N. (1995) Nature 377, 591-594.
8. Wen, Z. et al. (1995) Cell 82, 241-250.
9. Yokogami, K. et al. (2000) Curr. Biol. 10, 47-50.
10. Biethahn, S. et al. (1999) Exp. Hematol. 27, 885-894.

Application References

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Companion Products

• 9138 Phospho-Stat3 (Tyr705) (3E2) Mouse mAb
• 7076 Anti-mouse IgG, HRP-linked Antibody
• 9803 Cell Lysis Buffer (10X)

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.