Edc nhs

Biacore surface plasmon resonance experiments were performed at 25 ° C on a Biacore 3000 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). CC1 sensor surface was prepared using ligand thiol coupling, with the cysteine residue at the N terminus of CGG-CC1(233–342) (ref. 6 (link)). The immobilization was performed on a CM3 sensor chip at 5 μl min⁻¹ flow rate with PBS (10 mM sodium phosphate pH 7.4, 150 mM NaCl). Flow cell was activated with 15 μl of EDC/NHS followed by 30 μl PDEA (GE Healthcare) to introduce a reactive disulfide group on the surface. Thirty microlitre of CC1 (10 μg ml⁻¹ in 10 mM sodium acetate pH 5.5) was injected onto the activated surface and then blocked with 30 μl of cysteine-NaCl. Approximately 650 response units of CC1 were immobilized. A reference flow cell was prepared with activation and blocking steps, but without any protein coupled. Binding study was carried out at 50 μl min⁻¹ flow rate using TBS (20 mM Tris, pH 7.0, 150 mM NaCl) containing 0.1% BSA (w/v) as running buffer. MBP-SOAR (0–16 μM, 2 × dilution) or MBP stock in TBS was first diluted twofold in TBS containing 0.2% BSA, then further diluted with running buffer and injected onto CC1 and reference surfaces. Background-corrected sensorgrams were collected and analysed using BIAevaluation (version 4.1).

Measurements were performed on a Biacore™ T200 (GE-Healthcare Bio-Sciences AB) using CM5 chips, and running buffer HBSP (10 mM Hepes, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at 25°C. Peptides and proteins were immobilized via amine coupling with EDC/NHS at a flow rate of 10 μl min^-1, according to the manufacturer’s instructions (GE Healthcare). Optimum preconcentration was evaluated before, using acetate and phosphate buffers (10 mM) of pH 4 to 7, and ligand concentrations of 10 μg ml^-1. For the immobilization of biotinylated sialyllactose (Lectinity), neutravidin (Thermo Fisher Scientific) (100 μg ml^-1) was immobilized by amine coupling at pH 5.5 enabling high ligand density. Biotinylated sialyllactose was then injected at a concentration of 10 μg ml^-1 in HBSP for 3 min.