The mAb cetuximab (Erbitux; 5 mg/mL) was purchased from Merck (Darmstadt, Germany) and was buffer exchanged on a PD10 column (GE Healthcare Life Sciences, Eindhoven, The Netherlands) to a solution of 0.9% NaCl before radiolabelling.
Eluted proteins were buffer exchanged on a C18 column to 20%acetonitrile+0.088% TFA and lyophilized.
The eluent was concentrated with an Amicon Ultra centrifugal filter, and buffer exchanged on a PD-10 desalting column.
Human PDI (PDIA1 18–508) and roGFP variants were expressed in the E. coli BL21 (DE3) strain, purified with Ni-NTA affinity chromatography, dialyzed into the reaction buffer, reduced by incubation with 20 mM of DTT, and then buffer exchanged on a PD-10 gel filtration column (GE Healthcare), as described previously (Avezov et al., 2013).
After elution, proteins were further purified and buffer exchanged on a Superdex-200 (10/30) gel filtration column in a solution of 20 mM CHES, pH 9.0, 150 mM KSCN, 0.1 mg/ml E. coli polar lipid, 2 mM DTT and 4 mM DM for the full-length GsuK channel, and in a solution of 50 mM Tris HCl, pH 8.0, 250 mM KCl, 2 mM DTT and 4 mM LDAO for the GsuK intracellular subunit.
Protein was eluted with 25 mM Tris pH 8.0, 200 mM NaCl, 1 M MgCl2, 10% glycerol, and 2% β-octylglucoside, buffer exchanged on a desalting column into 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 10% glycerol, and 2% β-octylglucoside, and loaded onto a monoQ column equilibrated in 10 mM Tris pH 8.0, 1 mM EDTA, 0.1 mM PMSF, 2.5% glycerol, and 0.03% Triton X-100.
When cetuximab was used, it was buffer-exchanged on a PD10 column (GE Healthcare Life Sciences, Eindhoven, The Netherlands) to a solution of 0.9% NaCl before chelate conjugation.
The monoclonal antibody (mAb) cetuximab (Erbitux; 5 mg/mL) was purchased from Merck (Darmstadt, Germany) and, before any chemical modification, cetuximab was buffer-exchanged on a PD10 column (GE Healthcare Life Sciences, Eindhoven, The Netherlands) to a solution of 0.9% NaCl.
CtCel6A was purified on a DEAE-Sepharose column after buffer exchange on a Toyopearl HW-40 column.
CBHs were purified on a Q-Sepharose column after buffer exchange on a Toyopearl HW-40 column.
The affinity-purified protein was eluted in presence of 250 mM imidazole and subjected to buffer exchange on a PD-10 column against the assay buffer.
