Revvity, 64IRK4T45PEG, HTRF Human Phospho-IRAK4 (Thr345) Detection Kit, 500 Assay Points

Overview
IRAK4 is a protein kinase belonging to the interleukin-1 receptor associated pathway (IRAKs) that plays a critical role in initiating innate immune response against pathogens.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
Phospho-IRAK4 (Thr345) assay principle

The Phospho-IRAK4 (Thr345) assay measures IRAK4 when phosphorylated at Thr345. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation leads to an immune-complex formation involving both labeled antibodies, which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-IRAK4 (Thr345) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-IRAK4 (Thr345) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored. Application-Cell Signaling
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-IRAK4
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology Rare Diseases
Unit Size-500 assay points