Product Pathways - PathScan ELISA
PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285
| Kit Includes | Volume | Solution Color |
|---|---|---|
| PTEN Antibody Coated Microwell | 96 tests | |
| PTEN (Ser380) 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 M Mk
Reactivity Key: H=Human M=Mouse Mk=Monkey
Description
CST's PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-PTEN (Ser380) protein. A PTEN Mouse mAb* has been coated onto the microwells. After incubation with cell lysates, PTEN protein (phospho and non-phospho) is captured by the coated antibody. Following extensive washing, Phospho-PTEN (Ser380) Antibody* is added to detect the captured phospho-PTEN (Ser380) protein. Anti-rabbit IgG, HRP-linked Antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of phospho-PTEN (Ser380) protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit detects endogenous levels of phospho-PTEN (Ser380) protein. As shown in Figure 1 using the Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285 a high level of phospho-PTEN (Ser380) is detected in HeLa cells. These levels are significantly reduced in HeLa cell lysates treated with λ phosphatase.
Sandwich ELISA
Figure 1. Phosphorylation of PTEN at Ser380 detected by PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285. PTEN in HeLa cells is constitutively phosphorylated at Ser380. Treatment of HeLa cell lysates with λ phosphatase (4000 U/mL for 60 minutes at 37°C) abolishes the phosphorylation of PTEN at Ser380 as shown using PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit and by Western analysis. The level of total PTEN detected by Western analysis remains unchanged. Absorbance450 nm is shown in the top figure while the corresponding Western blots using Phospho-PTEN (Ser380) Antibody #9551 (right panel) or PTEN Antibody #9552 (left panel) are shown in the bottom figure.
Sandwich ELISA
Figure 2. The relationship between the protein concentration of untreated and λ phosphatase-treated HeLa cell lysates (see the legend of Figure 1) and the absorbance at 450 nm is shown.
Background
PTEN (phosphatase and tensin homologue deleted on chromosome ten), also referred to as MMAC (mutated in multiple advanced cancers) phosphatase, is a tumor suppressor implicated in a wide variety of human cancers (1). PTEN encodes a 403 amino acid polypeptide originally described as a dual-specificity protein phosphatase (2). The main substrates of PTEN are inositol phospholipids generated by the activation of the phosphoinositide 3-kinase (PI3K) (3). PTEN is a major negative regulator of the PI3K/Akt signaling pathway (1,4,5). PTEN possesses a carboxy-terminal, noncatalytic regulatory domain with three phosphorylation sites (Ser380, Thr382 and Thr383) that regulate PTEN stability and may affect its biological activity (6,7). PTEN regulates p53 protein level and activity (8) and is involved in G protein coupled signaling during chemotaxis (9,10).
- Cantley, L.C. and Neel, B.G. (1999) Proc. Natl. Acad. Sci. USA 96, 4240-4245.
- Myers, M.P. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 9052-9057.
- Myers, M.P. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 13513-13518.
- Wan, X. and Helman, L.J. (2003) Oncogene 22, 8205-8211.
- Wu, X. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 15587-15591.
- Vazquez, F. et al. (2000) Mol. Cell. Biol. 20, 5010-5018.
- Torres, J. and Pulido, R. (2001) J. Biol. Chem. 276, 993-998.
- Freeman, D.J. et al. (2003) Cancer Cell 3, 117-130.
- Funamoto, S. et al. (2002) Cell 109, 611-623.
- Iijima, M. and Devreotes, P. (2002) Cell 109, 599-610.
Application References
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