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| | Surface Plasmon Resonance Assay |
| Description: | Using a Biacore T200, Avidin tagged VHL co-expressed with Elongins B and C were immobilized to a Biacore SA chip in running buffer without DMSO. Compounds were tested individually at varying concentrations in running buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.5 mM TCEP, 0.001% Tween 20, 0.2% PEG3350, 2% DMSO) at 20° C. Sensorgrams were run in order from low to high concentration using a flow rate of 80 μL/min. Association and disassociation times were varied depending on the estimated potency of the compound tested. All sensor chips were monitored for loss of activity with the injection of a control compound ((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, which is Compound 7 in C. Galdeano et al, J. Med. Chem. 2014, 57, 8657-8663, herein incorporated by reference) which retained greater than 75% of the activity over the course of the run. Analysis of the binding curves and determination of the kinetic parameters were done using evaluation software (Version 2.0, Biacore). |
| Affinity data for this assay | |
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