Cell Signaling Technology

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

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).

Sandwich ELISA

Sandwich ELISA

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.

Sandwich ELISA

Sandwich ELISA

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 Stat3 rabbit monoclonal antibody (7300-52F7) 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 rabbit monoclonal antibody (7300-52F7), is shown in the bottom figure.

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.

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Companion Products

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

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