cGMP for accurate assessment in cells and for PDE
The Total eNOS kit is designed to monitor the expression level of cellular eNOS (endothelial nitric oxide synthase), and can be used as a normalization assay for the Phospho-eNOS (Ser1177) kit.
The Total eNOS cellular assay monitors total eNOS, and can be used as a normalization assay with our phospho-eNOS (S1177) kit. This kit is compatible with the buffers from the phospho-eNOS kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
eNOS is expressed primarily in endothelial cells, where its function is to control blood vessel dilation, blood pressure, and numerous other anti-atherosclerotic and vasoprotective effects.
Serine 1177 is the most important phosphorylation site of eNOS, positively modulating its activity. Phosphorylation at serine 1177 is under the control of several kinases, including AKT, PKA, AMPK, CaMKK2b, and CHKI.
The Total eNOS assay quantifies the expression level of eNOS in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total eNOS assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of eNOS in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.
Huvec cells were seeded in a 160mm Petri Dish at 80% of confluence in 37°C, 5% CO2 conditions, and an overnight serum starvation was done or not. Then the addition of a solution of pervanadate at 30µM for 30 minutes was done or not. Lysis buffer was added to each dish after culture medium removal, and incubated for 30 minutes under shaking. Next, 16µl of lysate were transferred into a 384 well plate for Total and Phospho Ser1177 eNOS detection.
Starvation seems to induce just a slight decrease in protein, but a real decrease in the phosphorylation level. Phosphorylation accumulation with pervanadate is efficient.
Huvec cells were seeded in a 100mm Petri Dish at 80% of confluence in 37°C, 5% Co2 conditions, and an overnight serum starvation was done. Then a fenofibrate solution at 30µM was added for 30 minutes or not. Lysis buffer was dispensed after culture medium removal. 16µl of lysate were transferred into a 384 well plate for Total and Phospho Ser1177 eNOS detection.
Huvec cells were grown in a 160mm Petri dish 37°C, 5% Co2, for 2 days. Overnight serum starvation was run before an incubation with pervanadate at 30µM for 30 min. After removal of cell culture medium, 3 mL of supplemented lysis buffer were added and incubated for 30 min. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. The Total-eNOS assay shows a better sensitivity than Western Blot: 37,500 cells/mL are sufficient to be detected by HTRF, while WB needs 75,000 .
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HTRF and WB compatible guidelines - Technical Notes
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Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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Detailed protocol and direct comparison with WB - Posters
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Analysis of a large panel of diverse biological samples and cellular models - Posters
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