PathHunter® Protein Degradation Assays

Monitor responses to pathway stimulation at the receptor level with PathHunter Degradation Assays.  These assays allow for profiling of small molecules or biologics that induce degradation of key signaling nodes (e.g. NIK or IƙB) through checkpoint or signaling pathways in one simple platform.

Features & Benefits

  • High-throughput platform to study protein degradation
  • Simple two-step assay
  • Profile multiple compound induced degradation in one simple platform

Technology Principle

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.

Analyze Stimulation of the NFkB Pathway Through IkB Degradation

The NF-κB pathway was stimulated with TNFα using the PathHunter A549 IκB Degradation Cell Line to measure IκB degradation. TNFα treatment leads to robust degradation of IκB and thus the cell line, similar to the other PathHunter IκB degradation cell lines, can be used to analyze therapeutics that stimulate or inhibit the NFκB pathway.