The phospho-IRF3 (Ser386) assay enables the cell-based detection of Ser386 phosphorylation on IRF3.
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  • No-wash No-wash
  • Low sample consumption Low sample consumption
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The phospho-IRF3 (Ser386) assay enables the cell-based detection of Ser386 phosphorylation on IRF3.
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Overview

The phospho-IRF3 cell based assay kit conveniently and accurately quantifies phosphorylated IRF3 at Ser386. In viral infected cells, phosphorylation of transcription factor IRF3 induces the production of antiviral and proinflammatory cytokines, a key element in the innate immunity system. This phospho-IRF3 assay can be used from basic research through to preclinical drug discovery phases and contains everything you need. It offers increased throughput compared to ELISA/WB.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-IRF3 (Ser386) assay principle

The phospho-IRF3 (Ser386) assay measures IRF3 when phosphorylated at Ser386. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-IRF3 (Ser386) 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 both labeled antibodies and which 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.
Phospho-IRF3 (Ser386) assay principle

Phospho-IRF3 (Ser386) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-IRF3 (Ser386) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-IRF3 (Ser386) 2-plate assay protocol

Phospho-IRF3 (Ser386) 1-plate assay protocol

Detection of Phosphorylated IRF3 (Ser386) 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.
Phospho-IRF3 (Ser386) 1-plate assay protocol

Optimization of Calyculin incubation time

Calyculin, a potent serine/threonine protein phosphatase inhibitor, was used to induce phosphorylation of IRF3 in MCF7 cells. After treatment for various incubation times (45 minutes, 1 hour, 2 hours, or 3 hours) with 400 nM Calyculin, MCF7 cells were lysed with 50 µL of supplemented lysis buffer and incubated for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-IRF3 detection reagents. The HTRF signal was recorded after an overnight incubation.
Optimization of Calyculin incubation time in MCF7 stimulated cells

IRF3 phosphorylation upon 2-3 cGAMP stimulation

125,000 MCF7 cells per well were pated in a 96-well pate in complete culture medium and incubated for 24h at 37°C, 5% CO2. 50 µg/mL of 2-3 cGAMP was added and then incubated for various incubation time at 37°C. After cell culture removal the cells were lysed with 50 µL of lysis buffer for 30 min at RT, 16 µL of lysate were then transferred into a 384-well sv white pate and 4 µL of the HTRF phospho-IRF3 detection reagents were added. The HTRF signal was recovered after over-night incubation.
IRF3 phosphorylation upon 2-3 cGAMP stimulation

Phospho-IRF3 assay compared to western Blot in human MCF7 cells

Human MCF7 cells were cultured for 48 hours and then stimulated with 200nM calyculin for 2 hours lysing. Soluble fractions were collected via centrifugation and serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. By using HTRF phospho-IRF3 (Ser386) only 2,500 cells are sufficient for minimal signal detection while 10,000 cells are needed for a Western Blot signal. The HTRF phospho-IRF3 assay is at least 4-fold more sensitive than the Western-blot.
Phospho-IRF3 assay compared to western Blot with human MCF7 cells

IRF3 Simplified Pathway.

IRF3 is a key transcriptional regulator of type I interferon (IFN-alpha and IFN-beta)-dependent immune responses. It plays a critical role in the innate immune response against DNA and RNA viruses. Found in an inactive form in the cytoplasm of uninfected cells, IRF3 becomes phosphorylated at Ser 386 by TBK1, following viral infection, double-stranded RNA (dsRNA), Toll-like receptor (TLR) and STING signaling pathways. This induces a conformational change, leading to its dimerization and nuclear localization where it can activate type I IFN gene expression.

IRF3 signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

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

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical 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

Unmatched ease of use, sensitivity and specificity assays - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Product Insert IRF3 P-386 Kit / 6FRF3PEG-6FRF3PEH

6FRF3PEG-6FRF3PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Novel HTRF platform to delineate STING pathway

Explore the whole STING pathway with a single technology - Posters

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

STING, the next candidate for cancer immunotherapy

Infographic about STING protein - Infographics

A Guide To Current Therapies in Immuno Oncology

Clear overview of past, present and future of immunotherapy - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

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

Characterize STING agonists with HTRF human IFNb

Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes

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