| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) |
| Description: | To assess the interaction between DDB1-BPB and compounds, a multi-parametric SPR instrument (BioRad ProteON XPR360) was used. DDB1-BPB was immobilized onto the GLH sensor surface in a variety of ways as follows: flow channels were activated in parallel with 1/20 diluted EDC/SNHS. DDB1-BPB was diluted to 0.1 mg/mL in sodium acetate pH 4.5 and directly coupled onto channel for 4 min. Excess reactive esters were blocked with a 5-min injection of 1 M ethanolamine. Mean immobilization levels were 17,000 RU with <2% variation along a strip. One channel was left unmodified to provide an additional reference surface. Next, the binding kinetics of compounds on surfaces was determined in a single injection using a “one-shot” kinetic mode. Typically, compound was prepared as a twofold serial dilution (0, 3.125, 6.25, 12.5, 25, 50 μM) and injected for 60 s at 100 L/min. Dissociation was monitored for 800 s. Surfaces were regenerated with multiple 30-s pulses of 60 mM phosphoric acid so that the experiment could be reproduced. |
| Affinity data for this assay | |
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