| Assay Method Information | |
| | Surface Plasmon Resonance (SP1R) |
| Description: | As the device, Biacore T200 (manufactured by GE Healthcare) was used. VHL was obtained by co-expressing Elongin B and Elongin C with a Twin-Strep-tag added to the N-terminus. As the sensor chip, Sensor chip CM5 (manufactured by GE Healthcare) was used. First, Strep-Tactin XT (manufactured by GE Healthcare) was immobilized on the sensor chip by amine coupling. Thereafter, VHL was immobilized using the affinity of Strep-Tactin XT and Twin-Strep-tag, and VHL was further immobilized on the sensor chip by amine coupling. As the running buffer, a mixed solution of 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, and 2% DMSO was used. Solutions of various compound concentrations of the test compounds (compounds described in Examples 1 to 162 above) were flowed on the sensor chip with VHL immobilized thereon at a flow rate of 30 uL/min under 25° C. conditions, and changes in mass on the sensor chip were observed. The Contact time and the Dissociation time were determined according to the properties of the compounds. For many compounds, 60 seconds and 120 seconds were respectively used. The observed data was analyzed using Biacore T200 Evaluation software Version 2.0 (manufactured by GE Healthcare) and the Kd value for VHL of each compound was calculated. |
| Affinity data for this assay | |
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