Phospho-eNOS (Ser1177) Cellular Kit HTRF®

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This HTRF kit enables the cell-based quantitative detection of phosphorylated eNOS (endothelial nitric oxide synthase) at Serine 1177.

No-wash
All inclusive kit
High sensitivity
Low sample consumption

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This HTRF kit enables the cell-based quantitative detection of phosphorylated eNOS (endothelial nitric oxide synthase) at Serine 1177.

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Overview

This HTRF cell-based assay conveniently and accurately detects phosphorylated eNOS at Serine 1177.

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

This HTRF cell-based assay conveniently and accurately detects phosphorylated eNOS at Serine 1177.

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Phospho-eNOS (Ser1177) assay principle

The Phospho-eNOS (S1177) assay measures eNOS when phosphorylated at serine 1177. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Principle of the HTRF phospho-eNOS (Ser1177) assay

Phospho-eNOS (Ser1177) 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 Phospho-eNOS (Ser1177) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Phspho-eNOS (Ser1177) assay

Phospho-eNOS (Ser1177) one-plate assay protocol

Detection of Phosphorylated eNOS (Ser1177) 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.

One-plate protocol of the HTRF Phspho-eNOS (Ser1177) assay

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 a solution of pervanadate at 30µM for 30 minutes was added or not. Lysis buffer was added to each dish after culture medium removal, and incubated for 30 minutes under shaking. Then 16µl of lysate were transferred into a 384 well plate for Total and Phospho Ser1177 eNOS detection.

Starvation seems to induce a slight decrease in protein, but a real decrease in the phosphorylation level. Phosphorylation accumulation with pervanadate is efficient.

Serum starvation and phosphorylation accumulation

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 then 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 phospho-eNOS (Ser1177)

Huvec cells were grown in a 160mm Petri dish at 37°C, 5% Co2, for 2 days. Overnight serum starvation was carried out before an incubation of 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. Phospho-eNOS (Ser1177)assay shows a better sensitivity than Western Blot: 37,500 cells/mL are sufficient to be detected by HTRF, while WB needs 75,000.

Comparison of HTRF phospho-eNOS (Ser1177) kit with western blot

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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Insider Tips for successful sample treatment - Technical Notes

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HTRF and WB compatible guidelines - Technical Notes

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Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

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One technology across all samples - Application Notes

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HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

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In collaboration with Bayer - Scientific Presentations

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

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3' video to set up your Phospho assay - Videos

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Product Insert eNOS p-S1177 kit / 64ENOSS7PEG-64ENOSS7PEH

64ENOSS7PEG-64ENOSS7PEH - Product Insert

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

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Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Safety Data Sheet (DEU) eNOS p-S1177 kit / 64ENOSS7PEG