Revvity, 64KKATPEG, HTRF Human Total IκKα Detection Kit, 500 Assay Points

Overview
Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) also known as IKK1 is a protein kinase encoded by the CHUK human gene. IKKα is part of the IκB kinase complex (IKK) composed of the catalytic subunits IKKα and IKKβ and the regulatory subunit NF-κB essential modulator (NEMO, IKKγ).
The activation of the IKK complex depends on the phosphorylation of IKKβ (Ser177/180) or IKKα (Ser176/Ser 180) resulting in complex conformational change and kinase activation. IKKα is a key regulator of the NF-κB pathway downstream of several proinflammatory factors including interleukin-1, tumor necrosis factor alpha (TNFα) and toll-like receptor agonists. Although IKKα and IKKβ are highly homologous sharing over 50% of structural similarity they have different downstream substrates and non-overlapping functions.
How it works
Total IKKα assay principle
The Total IKKα assay quantifies the expression level of IKKα in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total IKKα assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of IKKα 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 IKKα 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 Total IKKα HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total IKKα one-plate assay protocol
Detection of Total IKKα 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 Lysis Buffer 4
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
Unit Size-500 assay points