Surface plasmon resonance (SPR) on a Biacore 8K instrument (Cytiva, Marlborough, MA, USA) was used to determine affinity and kinetics of sequestrins binding to Aβ1–40. Series S SA sensor chips (Cytiva, Marlborough, MA, USA) were immobilized with 120 response units (RU) of biotinylated Aβ1–40 (AnaSpec, Fremont, CA, USA) according to manufacturer’s instructions. PBST (0.05% Tween-20) was used as running buffer. All protein candidates were injected as a multi-cycle analysis in an 8-step 1:1.5 dilution series from 342 nM to 30 nM at 25 °C in duplicate. The dimeric affibody Ztaq3638-(G4S)2-Ztaq3638-ABD was used as negative control [18 (link)]. Analyte was injected for 200 s and dissociation was recorded for 600 s at 30 μL/min. Surfaces were regenerated with 10 mM HCl for 35 s and let to stabilize for 30 s before next cycle. The results were evaluated using the Multi-cycle kinetics method—1:1 binding, using the Biacore Insight Evaluation Software (Version 2.0.15, Cytiva, Marlborough, MA, USA).
In a second analysis, Series S SA sensor chips were immobilized with 80 response units (RU) of biotinylated Aβ1–40, and SqAβ22, and SqAβ23 were injected as analyte in a 1:2 dilution series ranging from 300 nM to 75 nM at 37 °C in duplicate.
In a second analysis, Series S SA sensor chips were immobilized with 80 response units (RU) of biotinylated Aβ1–40, and SqAβ22, and SqAβ23 were injected as analyte in a 1:2 dilution series ranging from 300 nM to 75 nM at 37 °C in duplicate.