Revvity, 64CGASTPEH, HTRF Human Total cGAS Detection Kit, 10,000 Assay Points

Overview
Following pathogen infection and the binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1, enabling its binding to IRF3 which leads to IFNs type 1 production. The STING pathway is then switched off by STING degradation, involving autophagy.
In immuno-oncology, activating the STING pathway has shown promising anti-tumor effects in pre-clinical models and thus represents a therapeutic strategy to treat human cancer.
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
Total cGAS assay principle
The Total cGAS assay quantifies the expression level of cGAS in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total cGAS assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of cGAS 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 cGAS two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total cGASdetection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-ATG16L1 one-plate assay protocol
Detection of Total cGAS 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
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
Unit Size-10,000 assay points