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HTRF Human Total cGAS Detection Kit HTRF®

The HTRF Human Total cGAS detection kit is designed to monitor the expression level of cellular cGAS.

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  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

The HTRF Human Total cGAS detection kit is designed to monitor the expression level of cellular cGAS.

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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.

Benefits

  • SPECIFICITY
  • PRECISION
  • LOW SAMPLE CONSUMPTION

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.

Principle of the HTRF (h) total cGAS assay

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.

2-plate protocol of the HTRF Human Total cGAS assay

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.

1-plate protocol of the HTRF Human Total cGAS assay

Validation of Total cGAS detection in siRNA-treated THP-1 cells

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.

Validation of Total cGAS detection in siRNA-treated THP-1 cells

Validation of Total cGAS detection in siRNA-treated HeLa cells

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.

Validation of Total cGAS detection in siRNA-treated HeLa cells

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Manual cGAS Total Kit / 64CGASTPEG-64CGASTPEH

64CGASTPEG-64CGASTPEH - Product Insert

Plate Reader Requirement

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.

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