This kit enables the detection of IRAK4 in lysates.

See more
  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
  • Enables fast assay qualification Enables fast assay qualification

This kit enables the detection of IRAK4 in lysates.

In stock


​IRAK4 (IL-1R-associated kinase 4) is a serine–threonine kinase essential in the signaling cascade associated with Toll-Like Receptors. IRAK4 is involved in the production of proinflammatory cytokines through NF-κB-mediated and mitogen-activated protein kinase (MAPK) activation, as well as IFNα/β secretion through interferon response factors 3 and 7 (IRF3/7).

This cell-based assay measures the modulation of IRAK4 protein levels.



Total IRAK4 assay principle

This total IRAK4 assay quantifies the expression level of IRAK4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total-IRAK4 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 IRAK4 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 total IRAK4

Total IRAK4 2-plate Assay protocol

​The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Total IRAK4 HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Total IRAK4 2-plate assay protocol

Total IRAK4 1-plate assay protocol

Detection of total IRAK4 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

Total IRAK4 1-plate assay protocol

Endogenous level of IRAK4 on THP-1 cells

THP-1 cells were plated in a 96-well plate (50 µL per well) and incubated for 15 min at 37°C, 5% CO2. Cells were then lysed with 50 µL of supplemented lysis buffer #1 at 2X for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Total IRAK4 detection reagents were added.

The HTRF signal was recorded after 4h of incubation.

Total IRAK4 on THP1 cells: cell density

IRAK4 is a master regulator of TLR/IL1R signaling

IRAK4 is a protein Kinase​ belonging to the interleukin-1 receptor-associated kinases (IRAKs), which are involved in signaling pathways tranduced by the Toll/Interleukin-1 receptor superfamily upon PAMPS recognition.

Like IRAK2 and IRAK1, IRAK4 signals through TRAF6, leading to the activation of the NFκB and MAPK pathways, and resulting in the transcription of pro-inflammatory cytokines.

Unlike other IRAK proteins, the suppression of IRAK4 activity completely impairs TLR receptor signaling, making it a pivotal component in the IL-1/TLR signaling pathway.

Simplified pathway for IRAK4 assays

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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

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

Cisbio lysis buffer compatibility

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

Species compatibility

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

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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

Insider Tips for successful sample treatment - Technical Notes

HTRF assays facilitate investigation of the TGFB/SMAD/a-SMA signaling axis in liver fibrosis

Benefits of using HTRF assays to characterize liver fibrosis - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Product Insert IRAK3 total kit / 63ADK101PEG-63ADK101PEH

63ADK101PEG-63ADK101PEH - Product Insert

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

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

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

Note about HMGB1 assay

HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes

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

Increased flexibility of phospho-assays - Application Notes

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

Product Insert IRAK4 total kit / 63ADK108PEG-63ADK108PEH

63ADK108PEG-63ADK108PEH - Product Insert

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

Tumor xenograft analysis: HTRF versus Western blot - Application Notes