Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285

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Kit Includes Volume Solution Color
PTEN Ab Coated Microwell 96 tests
PTEN (S380) DetectionAb 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M Mk

Reactivity Key:  H=Human  M=Mouse  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

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 37ºC) 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.

Sensitivity

Sensitivity

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 levels and activity (8) and is involved in G protein-coupled signaling during chemotaxis (9,10).

  1. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  2. Myers, M.P. et al. (1997) Proc Natl Acad Sci USA 94, 9052-7.
  3. Myers, M.P. et al. (1998) Proc Natl Acad Sci USA 95, 13513-8.
  4. Wan, X. and Helman, L.J. (2003) Oncogene 22, 8205-11.
  5. Wu, X. et al. (1998) Proc Natl Acad Sci USA 95, 15587-91.
  6. Vazquez, F. et al. (2000) Mol Cell Biol 20, 5010-8.
  7. Torres, J. and Pulido, R. (2001) J Biol Chem 276, 993-8.
  8. Freeman, D.J. et al. (2003) Cancer Cell 3, 117-30.
  9. Funamoto, S. et al. (2002) Cell 109, 611-23.
  10. Iijima, M. and Devreotes, P. (2002) Cell 109, 599-610.

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