Phospho-IRF3 (Ser386) Cellular Assay Kit

HTRF® cell-based assay for quantifying phosphorylated IRF3

Cisbio's cell-based homogeneous HTRF® phospho-IRF3 (Ser386) immunoassay enables the quantitative detection of IRF3 phosphorylated on Serine 386. In viral infected cells, the phosphorylation of transcription factor IRF3 induces the production of antiviral and proinflammatory cytokines, a key element in the innate immunity system. Using a streamlined protocol without any washing steps and amenable to low-volume formats, this phospho-IRF3 assay can be used from basic research through to preclinical drug discovery phases. The kit contains all the reagents you need, and offers increased throughput compared to ELISA or Western Blot.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio's phospho-IRF3 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-IRF3 antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-IRF3 antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The phospho-IRF3 assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, phospho-IRF3 can be quantitatively detected using the HTRF phospho-IRF3 kit reagents and most TR-FRET multimode plate readers.

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection of phosphorylated IRF3, lysates are subsequently transferred to a 384-well small volume assay plate, where the HTRF reagents are added. This also enables the monitoring of cell viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Product performances

HTRF assay detects the phosphorylation of IRF3 in Calyculin stimulated MCF7 cells

1.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.

Data shows that maximum signal is obained for Calyculin incubated for 2 hours.

2. 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.

3. HTRF assay compared to Western Blot on human MCF7 cells

Human MCF7 cells were grown in a T175 flask at 37°C, 5% CO2 for 48 hours. Cells were then stimulated with 200nM calyculin for 2 hours at 37°C. After medium removal, the cells were lysed with 3mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16µl 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.

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), or even toll-like receptor (TLR) signaling. This induces a conformational change, leading to its dimerization and nuclear localization where it can activate type I IFN gene expression.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
IRF3 phospho-S386 kit - 500 tests6FRF3PEG
IRF3 phospho-S386 kit - 10,000 tests6FRF3PEH
IRF3 phospho-S386 kit - 50,000 tests6FRF3PEY

Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein lysis buffer #2 - 130 mL64KL2FDF
IRF3 phospho-S386 kit control lysate6FRF3TDA