| Assay Method Information | |
| | Determination of TET2 Binding by Surface Plasmon Resonance |
| Description: | The binding of compounds of the examples was determined by Surface Plasmon Resonance (SPR) using a Biacore T200 SPR biosensor instrument (GE Healthcare, Uppsala, Sweden) at 25° C. The human TET2 construct was immobilized on a CM5 sensor chip using a standard covalent immobilization via amine coupling following the activation of the carboxymethyl dextran matrix of the sensor chip (injection of a solution containing 0.1 M N-hydroxysuccinimide and 0.4 M 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride at a flow rate of 15 μL/min for 7 min). The protein immobilization was achieved after the injection of 5 μg/mL TET2 in 10 mM sodium acetate (pH 4.0) at a flow rate of 5 μL/min. Unreacted activated groups of the dextran matrix were deactivated by injection of 1 M ethanolamine hydrochloride for 7 min at a flow rate of 15 μL/min. The corresponding matrix activation and protein immobilization were performed using as a running buffer a phosphate buffered saline solution (1.05× PBS: 10 mM phosphate, pH 7.4, 150 mM NaCl). The screened compounds were prepared in a 20 mM stock solution in DMSO and diluted with 1.05× PBS to achieve a final 5% (v/v) DMSO concentration. The running buffer of the interaction assays consisted of 1×PBS, 5% (v/v) DMSO. The flow rate used for the screening was 60 μL/min and the ligand association and dissociation times set were 60 s and 120 s, respectively. |
| Affinity data for this assay | |
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