| Assay Method Information | |
| | CYPA Binding Analysis by Surface Plasmon Resonance (SPR) Assay |
| Description: | All SPR experiments were conducted on a Biacore 8K+ instrument (Cytiva). Recombinant biotinylated CYPA protein with a C-terminal AVI tag was purified and immobilized on flow cell 2 of SA chip (Cytiva, BR100531), with the flow cell 1 as the blank reference. The running buffer was HBS-P+ Buffer (Cytiva, BR100827, 0.01 M HEPES pH 7.4,0.15 M NaCl, 0.05% v/v Surfactant P20), and the flow rate was 10 μL/min. The immobilization level was achieved to about 1800 response units (RU). Compounds were 3-fold serially diluted to 1.5-3333 nM using HBS-P+Buffer with a final DMSO concentration of 1.5%, and then loaded to flow through the chip surface at a rate of 30 μL/min with HBS-P+Buffer containing 1.5% DMSO as the running buffer. Experiments were performed at 25° C. The contact time and dissociation time were set to 60 s and 200 s, respectively. After binding and dissociation of each sample, the chip was regenerated using 1 M NaCl with a short contact time of 19 s at a rate of 60 μL/min. The sensorgrams were analyzed using Biacore Insight Evaluation software with 1:1 kinetics binding model to determine the association rate constant (kon), dissociation rate constant (koff) and equilibrium dissociation constant (KD). |
| Affinity data for this assay | |
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