Revvity, 64ENOSS7PEG, HTRF Human Phospho-eNOS (Ser1177) Detection Kit, 500 Assay Points

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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HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
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.

 

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.

 

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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Cardiovascular Metabolism/Diabetes
Unit Size-500 assay points