Total TBK1 cellular kit
All-in-one kit for robust detection of Total TBK1
Phospho-IRF3 (Ser386) cellular kit HTRF®
The phospho-IRF3 (Ser386) assay enables the cell-based detection of Ser386 phosphorylation on IRF3.
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No-wash
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Low sample consumption
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Faster and more convenient than ELISA
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 proteins phosphorylation state under a no-wash assay format.
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) 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.
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.
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.
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.
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.
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
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the Immune System - Brochures
PI3K/AKT/mTor translational control pathway - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
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
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
An innate and adaptive immunity recap
Insight into the diversity of cells & signaling pathways - Guides
HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes
Immuno-Oncology Drug Discovery
Explore our solutions for Immuno-oncology research - Flyers
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
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
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (ELL) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (ITA) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (SPA) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) IRF3 P-386 Kit / 6FRF3PEG
6FRF3PEG - Safety Data Sheet
Safety Data Sheet (DEU) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (ELL) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (ITA) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (SPA) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) IRF3 P-386 Kit / 6FRF3PEH
6FRF3PEH - Safety Data Sheet
Safety Data Sheet (DEU) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (ELL) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (ITA) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (SPA) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
Safety Data Sheet (ENG-US) IRF3 P-386 Kit / 6FRF3PEY
6FRF3PEY - Safety Data Sheet
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