Our VHL Cooperativity Assay Kit is a fluorescence polarization (FP)-based assay designed to evaluate the influence of a protein of interest (POI) on the apparent binding affinity of VHL-recruiting PROTACs for the VHL E3 ubiquitin ligase complex (VBC).

The assay uses CELT-504  as a high-affinity fluorescent tracer (low nanomolar range) and compares PROTAC displacement curves in the absence (binary condition) and presence (ternary condition) of the POI. This enables the determination of IC₅₀ and apparent Ki values, as well as the calculation of the cooperativity factor (α), providing insights into PROTAC-induced ternary complex formation. To perform the cooperativity assay, users must provide their own purified protein of interest (POI), as no POI is included in the kit. The selected protein should be suitable for the PROTAC being evaluated.

The assay has been validated using the well-characterized PROTAC MZ1 and BRD4-BD2 as the protein of interest, demonstrating strong positive cooperativity (α ≈ 23.6), consistent with published studies. In contrast, the monovalent VHL binder VH298 showed no positive cooperativity.

The homogeneous FP format requires no washing or separation steps and is suitable for comparative profiling of VHL-recruiting PROTAC candidates, supporting ternary complex characterization and targeted protein degradation research.

For more information on fluorescence polarization techniques, please refer to our blog article.