Glc chip

V_HH association and dissociation rates were determined by SPR using a ProteOn XPR36 system (Bio-Rad Inc., Hercules, CA, USA). Ricin was immobilized on a general layer compact (GLC) chip (Bio-Rad Inc.) equilibrated in PBS-0.005% Tween running buffer at a flow rate of 30 µL/min. Following EDAC [N-ethyl-N=-(3-dimethylaminopropyl) carbodiimide hydrochloride] (200 mM)–sulfo-NHS (N-hydroxysulfosuccinimide) (50 mM) activation (3 min), ricin was diluted in 10 mM sodium acetate (pH 5.0) at either 4 µg/mL or 2 µg/mL and coupled for 2 min. A third vertical channel received only acetate buffer and served as a reference channel. The surfaces were deactivated using 1 M ethanolamine for 5 min. A ProteOn array system multichannel module (MCM) was rotated to the horizontal orientation for affinity determination experiments. Each V_HH was serially diluted in running buffer and then injected at 50 µL/min for 180 s, followed by 1 to 3 h of dissociation. After each experiment, the chip was regenerated with 10 mM glycine (pH 1.5) at 100 µL/min for 18 s, until the response unit (RU) values had returned to baseline. All kinetic experiments were performed at 25 °C. Kinetic constants for the antibody/ricin interactions were obtained with ProteOn Manager software 3.1.0 (Bio-Rad Inc.) using the Langmuir fit model.

Protein-protein interactions were measured with a ProteOn XPR36 (Bio-Rad) in 10 mM Na-HEPES, 150 mM NaCl, 2 mM CaCl₂, and 0.05% Tween 20, pH 7.4 (running buffer) at 25°C. Proteins were immobilized on a GLC chip (Bio-Rad) via standard amine coupling with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDS)/N-hydroxysulfosuccinimide (s-NHS) using 10 µg/ml LOXL2 diluted in 100 mM acetate buffer, pH 5.0 at 100 µl/min for 60 s. Free reactive N-hydroxysulfosuccinimide (NHS) esters were blocked using ethanolamine. A reference lane was activated and blocked with ethanolamine but with no LOXL2 added. The chip was rotated by 90°, and analytes were flowed over the LOXL2 and reference sensors in sequential lanes using a serial 1:1 dilution from 830 nM TE at a flow rate of 100 µl for 120 s with a dissociation time of 300 s. Regeneration of the LOXL2 was achieved using a 30-s pulse of 50 mM NaOH.

The affinity and avidity of the anti-HER2 constructs for their substrate was measured at 25°C using a ProteOn XPR36 (BioRad). Fc-HER2 ectodomain produced in mammalian cells (96 kDa) was diluted to 2 μg/mL in sodium acetate buffer (pH 5.0) and immobilized by amine-coupling on a GLC chip (BioRad) at 2000 RU. 100 μL of antibodies were used as analyte and injected at 100 μL/min. The complete kinetic data set for each antibody was collected in a single run and fitted using a 1:1 Langmuir interaction model. Surface regeneration was performed using 10 mM glycine HCl, pH 2.0 (IgG-like format) or 3.0 (VHH format).

Experiments were performed on a ProteOn XPR36 instrument (BioRad Laboratories) using a running buffer containing 100 mM NaCl, 10 mM HEPES pH 7.0 and 0.1% (v/v) Igepal. Recombinant XRCC4-6×His was immobilised on a GLC chip (BioRad Laboratories) in the vertical orientation. Chip channels were activated using a mixture of 25 mM N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and 15 mM sulfo-N-hydroxysuccinimide (sulfo-NHS). Proteins were immobilised in 10 mM sodium acetate buffer, pH 4.5 (BioRad Laboratories). Remaining crosslinking sites were blocked by injection of 150 μl of 1 M ethanolamine–HCl (pH 8.5). Different SUMO or ubiquitin topologies were run in horizontal orientation at concentrations specified in each panel. Surface regeneration was accomplished with a pulse of 50 mM NaOH at 100 μl/min. All experiments were performed at 25°C. Regression curves were obtained via non-linear regression based on a single-site binding model (GraphPad Prism v6.0h).