Total IRAK4 cellular kit HTRF®

Name: Total IRAK4 cellular kit
Brand: Cisbio
SKU: 44495
Availability: InStock

Associated products

This kit enables the detection of IRAK4 in lysates.

All inclusive kit
High sensitivity
Enables fast assay qualification

Download the protocols

This kit enables the detection of IRAK4 in lysates.

Size

Cat. number selected :

Quantity

In stock

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.

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF assays facilitate investigation of the TGFB/SMAD/a-SMA signaling axis in liver fibrosis

Benefits of using HTRF assays to characterize liver fibrosis - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Product Insert IRAK3 total kit / 63ADK101PEG-63ADK101PEH

63ADK101PEG-63ADK101PEH - Product Insert

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

PI3K/AKT/mTor translational control pathway - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery

One technology across all samples - Application Notes

Note about HMGB1 assay

HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Scientific Presentations

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Product Insert IRAK4 total kit / 63ADK108PEG-63ADK108PEH

63ADK108PEG-63ADK108PEH - Product Insert

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

Assays for autoimmunity research

Advance your research on autoimmune diseases - Flyers

Advance your research on Fibrosis

Kits and reagents for Fibrosis research - Flyers

Safety Data Sheet (DEU) IRAK4 total kit / 63ADK108PEG