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Total IRAK3 cellular kit
This kit enables the detection of cellular IRAK3, and can be used as a normalization assay for the phospho-IRAK3 kit.
- All inclusive kit
- High sensitivity
- Enables fast assay qualification
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.
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.
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.
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
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.
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.
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