PathHunter Protein Degradation assays utilize the EFC cell-based assay format to detect target protein degradation, such as specific nodes in signaling pathways. The assay principle shown in the above example uses A549 cells that were engineered to express IκBα with the EFC small enzyme donor (ED) ProLabel tag attached to the C-terminus of IκBα. In response to TNFα, endogenous TNFα receptor present in the A549 cells signals through IκBα, resulting in IκBα protein degradation, a key step leading to NF-kB transcriptional activity. In this example, PathHunter A549 IκB Functional Assay is used with the PathHunter ProLabel /ProLink Detection Kit (Cat. No. 93-0812; which contains the large EFC enzyme acceptor (EA) necessary for the complementation to generate an active β-galactosidase enzyme) for measuring human IκB degradation in response to pathway stimulation.
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