Revvity, 64NFTPEG, HTRF Human and Mouse Total NF-κB Detection Kit, 500 Assay Points

Overview
Total NFkB kit serves as a normalization assay with our Phospho-NFkB kits. NFkB (Nuclear Factor Kappa B) is of great interest in cancer research and a key player in the inflammatory response. The buffers of both HTRF phospho- and total NFkB assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.
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-NFkB assay principle
The Total-NFkB assay quantifies the expression level of NFkB in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-NFkB 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 NFkB 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 proteins expression under a no-wash assay format.

 

Total-NFkB 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total NFkB HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-NFkB 1-plate assay protocol
Detection of total NFkB 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 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-NASH/Fibrosis Neuroscience Oncology & Inflammation
Unit Size-500 assay points