HTRF Human Total eNOS Detection Kit, 500 Assay Points

Total-eNOS assay principle

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.

Total eNOS two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total eNOS HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total eNOS one-plate assay protocol

Detection of Total eNOS with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Serum starvation and phosphorylation accumulation

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.

eNOS phosphorylation induced by fenofibrate stimulation through AMPK pathway

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.

WB versus HTRF assay for total-eNOS

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 .​​​​​​​