Revvity, 63ADK101PEH, HTRF Human Total IRAK3 Detection Kit, 10,000 Assay Points

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.
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
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 Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology & Inflammation
Unit Size-10,000 assay points