Revvity, 64KKAS6PEH, HTRF Human Phospho-IκKα (Ser176/180) Detection Kit, 10,000 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
Phospho-IKKα (Ser176/180) assay principle
The Phospho-IKKα (Ser176/180) assay measures IKKα when phosphorylated at Ser176 and Ser180. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-IKKα (Ser176/180) 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, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and 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-IKKα (Ser176/180) 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 adding Phospho-IKKα (Ser176/180) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-IKKα (Ser176/180) one-plate assay protocol
Detection of Phosphorylated IKKα (Ser176/180) 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 3 Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-10,000 assay points