This kit enables the detection of cellular IRAK3, and can be used as a normalization assay for the phospho-IRAK3 kit.

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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 cellular IRAK3, and can be used as a normalization assay for the phospho-IRAK3 kit.

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Overview

​IRAK3 is a mediator of toll-like receptor (TLR) and interleukin-1 receptor (IL1R). IRAK3 binds to MyD88 and to IRAK2/4, to inhibit the phosphorylation and activation of IRAK1. IRAK2 acts as a brake on TLR/ILR pathways and has a well-established role in regulating innate immunity. 

This cell-based assay measures the modulation of IRAK3 protein levels.

Benefits

  • SPECIFICITY
  • PRECISION

Total IRAK3 assay principle

The total IRAK3 assay quantifies the expression level of IRAK3 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total-IRAK3 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 IRAK3 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.

Total IRAK3 Assay principle

Total IRAK3 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 IRAK3 HTRF detection reagents.

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

Total IRAK3 2-plate Assay protocol

Total IRAK3 1-plate assay protocol

Detection of total IRAK3 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 IRAK3 1-plate assay protocol

Endogenous level of IRAK3 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 IRAK3 detection reagents were added.

The HTRF signal was recorded after 4h of incubation.

Endogenous level of IRAK3 on THP-1 cells

IRAK3 and its role as a negative regulator of TLR/IL1R signaling

​The interleukin-1 receptor-associated kinases (IRAKs) are mediators of toll-like receptors (TLR) and interleukin-1 receptor (IL1R) signaling. 

IRAK4, IRAK2 and IRAK1 signal through TRAF6, thus activating the NFκB and MAPK pathways, and resulting in the transcription of pro-inflammatory cytokines.

IRAK3 inhibits the TLR/IL1R pathway. IRAK3 prevents the dissociation of the active IRAKs (IRAK-1 and IRAK-4) from MyD88,  and in doing so plays the role of a negative regulator of innate TLR-mediated immune responses.

IRAK3 - IL1R/TLR Signaling pathway
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