Advanced phospho-ERK (Thr202/Tyr204) cellular kit
Simple, all-in-one kit for robust detection of Phospho-ERK.
Total IRAK3 cellular kit HTRF®
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
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High sensitivity
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Enables fast assay qualification
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 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 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
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.
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.
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
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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
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Insider Tips for successful sample treatment - Technical Notes
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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 2020 July update
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
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
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
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - 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
An innate and adaptive immunity recap
Insight into the diversity of cells & signaling pathways - Guides
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
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