Revvity, 64MSTGPEH, HTRF Mouse Phospho-STING (Ser365) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately quantifies mouse phosphorylated STING at Ser365. Following pathogen infection and binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1. This enables its binding to IRF3, which induces IFNs type 1 production and other immune responses. The STING pathway is then switched off by STING degradation involving autophagy.
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
Mouse Phospho-STING (Ser365) assay principle
The Mouse Phospho-STING (Ser365) assay measures mouse STING when phosphorylated at Ser365. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Mouse Phospho-STING (Ser365) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one 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 independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies. This 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.

 

Mouse Phospho-STING (Ser365) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Mouse Phospho-STING (Ser365) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Mouse Phospho-STING (Ser365) one-plate assay protocol
Detection of Mouse Phosphorylated STING (Ser365) 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 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Mouse
Technology-TR-FRET
Unit Size-10,000 assay points