Description
The total-IKK-beta assay enables the detection of total IKK-beta directly in whole cells. Using a streamlined protocol, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.
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
Total-IKKß assay principle
The Total IKK-beta assay quantifies the expression level of IKK-beta in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total IKK-beta 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 IKKß 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-IKKß 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 IKK-beta HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-IKKß cellular 1-plate assay protocol
Detection of total IKK-beta cellular 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.
Detection of phospho and total IKK-beta on HeLa cells
Different cell densities (200K and 100K) of HeLa cells were plated under 100 µL in 96-well plate and incubated overnight. Media was aspirated and 50 µL of different concentrations of IL-1 beta was added during 15 minutes. After incubation, media was aspirated and cells were lysed with 50 µL of lysis buffer 1X for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho IKK-beta (Ser177/181) or total IKK-beta detection reagents were added. The HTRF signal was recorded after a 2 hour incubation at room temperature.
IKKß Simplified Pathway
Activation of the NF-?B is initiated by the signal-induced degradation of I?B proteins. This occurs primarily via activation of a kinase called the I?B kinase (IKK). IKK is composed of a heterotrimer of 3 subunits, IKK-alpha and IKK-beta (the two catalytic subunits) and IKK-gamma/NEMO (a regulatory component). Activated IKK-beta phosphorylates a protein called the inhibitor of NF-?B, I?B (I?Ba), which binds NF-?B to inhibit its function. Phosphorylated I?B is degraded via the ubiquitination pathway, freeing NF-?B and allowing its entry into the nucleus of the cell, where it activates various genes involved in inflammation and other immune responses. IKK-beta plays a significant role in brain cells following a stroke.
