Total TBK1 cellular kit
All-in-one kit for robust detection of Total TBK1
The HTRF Human Total cGAS detection kit is designed to monitor the expression level of cellular cGAS.
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.
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.
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.
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.
THP-1 cells were plated at 100,000 cells per well under 90µl in a 96-well plates in complete culture medium. For Lipofectamine treated samples,10µl of Lipofectamine 1%f in culture medium were added. For siRNA treated cells, a 10µl mixture of Lipofectamine® RNAiMax/siRNA for cGas was added. Cells were then incubated for 48 h and 72 h at 37°C, 5% CO2.
After incubation, cells were lysed with 33.3µL of supplemented lysis buffer #1 at 4X for 30 minutes at RT under gentle shaking, and 16µL of lysate were transferred into a low volume white microplate before adding 2µL of the HTRF d2 detection reagent and2 µL HTRF Eu-K detection reagent. The HTRF signal was recorded after an ON incubation.
HELA cells were plated at 50,000 cells per well under 90µl in a 96-well plates in complete culture medium. For Lipofectamine treated samples,10µl of Lipofectamine 1%f in culture medium were added. For siRNA treated cells, a 10µl mixture of Lipofectamine® RNAiMax/siRNA for cGas was added. Cells were then incubated for 24 h and 48 h at 37°C, 5% CO2.
After incubation, cells were lysed with 90µL of supplemented lysis buffer #1 at 1X for 30 minutes at RT under gentle shaking, and 16µL of lysate were transferred into a low volume white microplate before adding 2µL of the HTRF d2 detection reagent and 2µL HTRF Eu-K detection reagent. The HTRF signal was recorded after an ON incubation.
Physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Learn how to reduce time and sample consumption - Application Notes
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.Let's find your reader