Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-eNOS (Ser1177) Sandwich ELISA Kit #7980

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Kit Includes Volume Solution Color
Phospho-eNOS Ab Coated Microwells 96 tests
eNOS Detection Ab 11 ml Green
Anti-mouse 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
ELISA 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

B

Reactivity Key:  B=Bovine
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-eNOS (Ser1177) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of eNOS when phosphorylated at Ser1177. A phospho-eNOS (Ser1177) rabbit monoclonal antibody has been coated onto the microwells. After incubation with cell lysates, phospho-eNOS protein is captured by the coated antibody. Following extensive washing, an eNOS mouse monoclonal detection antibody is added to detect captured eNOS protein phosphorylated at Ser1177. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate (TMB) is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of eNOS phosphorylated at Ser1177.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-eNOS (Ser1177) Sandwich ELISA Kit #7980 detects endogenous levels of eNOS protein only when phosphorylated at Ser1177 (see Figure 1). The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 2. The relationship between the protein concentration of lysates from BAEC cells treated with H2O2 or λ-phosphatase-treated lysate and the absorbance at 450 nm using PathScan® Phospho-eNOS (Ser1177) Sandwich ELISA Kit #7980 is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of BAEC cells with 500 µM of H2O2 increases phosphorylation of eNOS at Ser1177, detected by the PathScan® Phospho-eNOS (Ser1177) Sandwich ELISA Kit #7980. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using eNOS Antibody #9572 (left panel) or Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 (right panel) are shown in the bottom figure.

Background

Endothelial nitric-oxide synthase (eNOS) is an important enzyme in the cardiovascular system. It catalyzes the production of nitric oxide (NO), a key regulator of blood pressure, vascular remodeling, and angiogenesis (1,2). The activity of eNOS is regulated by phosphorylation at multiple sites. The two most thoroughly studied sites are the activation site Ser1177 and the inhibitory site Thr495 (3). Several protein kinases including Akt/PKB, PKA, and AMPK activate eNOS by phosphorylating Ser1177 in response to various stimuli (4,5). In contrast, bradykinin and H2O2 activate eNOS activity by promoting both Ser1177 phosphorylation and Thr495 dephosphorylation (6,7).

  1. Fulton, D. et al. (2001) J. Pharmacol. Exp. Ther. 299, 818-824.
  2. Shaul, P.W. (2002) Annu. Rev. Physiol. 64, 749-774.
  3. Chen, Z.P. et al. (1999) FEBS Lett. 443, 285-289.
  4. Dimmeler, S. et al. (1999) Nature 399, 601-605.
  5. Fulton, D. et al. (1999) Nature 399, 597-601.
  6. Harris, M.B. et al. (2001) J. Biol. Chem. 276, 16587-16591.
  7. Thomas, S.R. et al. (2002) J. Biol. Chem. 277, 6017-6024.

Application References

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