Superdex 200 5 150 column

The proteins and protein complexes were analyzed by analytical gel filtration using a Superdex 200 5/150 column (GE Healthcare) and the 1260 Infinity Bio-inert high-performance liquid chromatography system (Agilent Technologies) at 10 °C. The system and column were equilibrated in 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP and 30 μL of each sample was injected by an auto sampler. The system was run at 0.2 mL/min for 20 min and the elution profile was recorded by a UV detector.

Analytical size-exclusion chromatography experiments were performed on calibrated Superdex200 5/150 column (GE Healthcare). All samples were eluted under isocratic conditions at 4°C in size-exclusion chromatography buffer (20 mM Tris, 150 mM NaCl, 1 mM TCEP) at a flow rate of 0.2 ml min⁻¹. Elution of proteins was monitored at 280 nm. The loading volume for each injection was 50 µl. In order to detect complex formation, proteins were mixed at 1 : 1 (molar ratio) and incubated for 2 h on ice. SDS–PAGE, followed by Coomassie staining, was used to detect proteins.

Molecular weight determination of zebrafish CA VI–PTX was performed using a Malvern Zetasizer μV instrument (Malvern Instruments Ltd., Worcestershire, UK) running static light scattering (SLS) and dynamic light scattering (DLS) methods. Analysis was performed using a liquid chromatography instrument (CBM-20A, Shimadzu Corporation, Kyoto, Japan) equipped with autosampler (SIL-20A), UV–VIS (SPD-20A) and fluorescence detector (RF-20Axs). Data were processed using Lab Solution Version 5.51 (Shimadzu Corporation) and OmniSec 4.7 (Malvern Instruments Ltd., Worcestershire, UK) softwares. A sample of the protein (50 μg) was injected on a Superdex 200 5/150 column (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM NaH₃PO₄, 500 mM NaCl pH 8 buffer. Runs were performed with flow rate of 0.1 ml/min at 20 °C using a thermostated cabin. The MW of the zebrafish CA VI–PTX was determined either by using a standard curve based on MW standard proteins (SEC analysis; CA 29 kDa, alcohol dehydrogenase 150 kDa, β-amylase 200 kDa, BSA 66 kDa, Sigma-Aldrich, Inc., St. Louis, MO, USA) or by calibrating the light scattering detector using the monomeric peak of BSA and light-scattering intensity (SLS).

Small-angle X-ray scattering measurements of scFv 2A2 at 2.1, 4.2, and 8.5 mg/ml were performed at the SWING beamline of the French national synchrotron facility (SOLEIL). Data was collected at 15 °C, a wavelength of 1.0332 Å and a sample-to-detector distance of 1.99 m. For batch measurements, the scattering from the buffer alone was measured before and after each sample measurement and was used for background subtraction with PRIMUS from the ATSAS package^{42 (link)}. For SEC-SAXS measurements, scFv 2A2 samples at 8.5 mg/ml were loaded onto a Superdex200 5/150 column (GE Healthcare) previously equilibrated in 50 mM HEPES pH 7.5 and 150 mM NaCl with or without addition of 5 mM CoCl₂. The measured SAXS images were normalized to the transmitted intensity and azimuthally averaged by using the in-house software Foxtrot (https://www.synchrotron-soleil.fr/en/beamlines/swing). The resulting SAXS curves were analyzed using CHROMIXS^{43 (link)} and PRIMUS^{44 (link)}.
Additional SAXS measurements were performed on beamline BM29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Samples were kept at 20 °C and data were collected at a wavelength of 0.0995 nm and a sample-to-detector distance of 1 m. 1D scattering profiles were generated and buffer subtraction was carried out by the automated data processing pipeline available at BM29.