Revvity, 64ENOSTPEH, HTRF Human Total eNOS Detection Kit, 10,000 Assay Points

Overview
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.
How it works
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. 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-Total
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-10,000 assay points