Capture-stabilize approach for membrane protein SPR assays

Chu, Ruiyin; Reczek, David; Brondyk, William

Conventional peptide-based Biacore assay. A mouse antibody capturing kit from GE was used for Biacore CM5 chip surface preparation. Following standard protocols provided by the manufacturer, the rabbit anti-mouse polyclonal antibody (GE) was first immobilized on the chip surface via amine coupling to the free carboxyl groups on the CM5 chip surface using standard NHS/EDC procedures. Mouse monoclonal antibodies against hCXCR5 clones 16D7, 79E7 and MAB190 (R&D Systems) were then captured by the anti-mouse antibody. Next, the hCXCR5 N-terminal peptide (MNYPLTLEMD LENLEDLFWE LDRLDNYNDT SLVENHLCPA TEGPLMASFK AVFVP) was injected over the captured anti-hCXCR5 antibodies at various concentrations to generate kinetics sensorgrams.