| Assay Method Information | |
| | SPR Assay |
| Description: | Surface Plasmon Resonance (SPR) assays have been developed to test the affinity (KD) of VHL or CRBN-based compounds to respective recombinant ligase complex/domain.The assays are based on surface plasmon resonance (SPR), which enables to measure the changes of the local refractive index due to changes of molecular mass on a gold chip surface in the case of a binding event and in a flowing system. To detect binding between both partners, the respective E3 ligase is immobilized to the chip surface, while the test compounds are flown over the chip surface at a steady velocity. The detected changes in the RU response are indicative of the binding event and are concentration dependent.For the SPR experiments, either a commercially available VHL complex (Merck, 23-044; composed of 5 units: VHL, Elongin B, Elongin C, Cul2, and Rbx1) exhibiting a his-tag at the Cul2 subunit or an internally produced biotin-tagged mouse CRBN thalidomide binding domain (mCRBN-TBD) was used. These tags provide the anchor for the capturing process to either an NTA or Streptavidin coated chip surface (immobilization level of 3,000-5,000 RU). Because of the rather complex structure of the VHL complex, it was additionally coupled to the chip surface by amino coupling to prevent any protein loss by disruption of the complex in the flowing system.To detect binding of compounds and extract dissociation constants KD for the tested compounds to the immobilized E3 ligase, concentration response curves of the compounds were recorded. Compounds were usually tested in 10-pt dilutions up to 20 μM final concentration in assay buffer and were flown over the chip at 30 μL/min. The contact time for each cycle includes 90 s for association and 200 s for dissociation of compounds. Every test cycle was read out as a sensorgram that was referenced to the sensor surface that does not present the target protein. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |