Advanced phospho-ERK (Thr202/Tyr204) cellular kit
Simple, all-in-one kit for robust detection of Phospho-ERK.
Total IRAK4 cellular kit
This kit enables the detection of IRAK4 in lysates.
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All inclusive kit
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High sensitivity
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Enables fast assay qualification
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.
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 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
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.
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.
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63ADK108PEG-63ADK108PEH - Product Insert
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