Total IRAK4 cellular kit

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.

​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.

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

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.

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.