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.
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.
|Product Includes||Volume||Solution Color|
|Phospho-eNOS Rabbit mAb Coated Microwells||96 tests|
|eNOS Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
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).
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