| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) Assay |
| Description: | The kinetic and affinity parameters of protein-exemplary compound interactions were evaluated by SPR. hsTEAD1 (209-426) was immobilized onto a CM7 (Series S) sensor chip via the standard amine coupling procedure, at 15° C. Prior to immobilization, the carboxymethylated surface of the chip was activated with 400 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 100 mM N-hydroxysuccinimide for 10 min. hsTEAD1 (209-426) was diluted to 11 μg/mL in 10 mM Bis-Tris at pH 6.5 and immobilized on the activated surface chip for 8 and 10 min, in order to reach 6000 to 9000 response units (RU). The remaining activated carboxymethylated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5 after which the surface chip was washed with 0.5% (w/v) sodium dodecyl sulphate and 50 mM glycine. HBS-N, which consists of 10 mM HEPES pH 7.4 and 150 mM NaCl, was used as the background buffer during immobilization. Exemplary compounds were prediluted in DMSO, diluted 1:50 in running buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.1 mM EGTA, 0.05% CHAPS) and injected at ten different concentrations using two-fold dilution series, from 100 μM to 0.2 μM. A DMSO solvent correction (1%-3%) was performed to account for variations in bulk signal and to achieve high-quality data. Interaction analysis cycles consisted of a 240 sec sample injection (30 μL/min; association phase) followed by 600 sec of buffer flow (dissociation phase). |
| Affinity data for this assay | |
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