This kit enables the detection of IRAK4 in lysates.

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  • 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.

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Overview

​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.

Benefits

  • SPECIFICITY
  • PRECISION

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
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