Ktatm purifier chromatography system

Cell-free supernatants were loaded onto a 1-ml HiTrap^TM MabSelect SuRe^TM Protein A column pre-equilibrated in 0.2 m citrate/phosphate buffer, pH 6, and operated on an ÄKTA^TM purifier chromatography system (both from GE Healthcare Europe GmbH) at a flow rate of 1 ml/min. Running buffer was 0.2 m citrate/phosphate buffer, pH 6. Wash buffer was 0.2 m citrate/phosphate buffer, pH 5. Heterodimer elution was performed using 20 mm sodium acetate buffer, pH 4.1, although homodimeric species were unbound (IgG-like homodimers) or eluted with 0.1 m glycine, pH 3 (scFv-Fc homodimers). Elution was followed by absorbance reading at 280 nm; relevant fractions were pooled and neutralized with 0.1 volume of 1 m Tris-HCl, pH 8. Fractions were analyzed under non-reduced conditions by SDS-PAGE.

P3 membranes were solubilized as described in the previous section and 200 µL of the detergent-solubilized supernatant was injected in a Superose 6 10/300 GL gel-filtration column (GE healthcare) equilibrated with 20 mM Tris-HCl pH 7.8, 150 mM NaCl, 10% (v/v) Glycerol, 0.1 mg/mL DDM, 0.02 mg/mL CHS. Fluorescence-detection size-exclusion chromatography (FSEC) experiments were performed in an ÄKTA^TM purifier chromatography system (GE healthcare) with a fluorescence detector (FP-4025, JASCO) connected “in line” with the column. The excitation and emission wavelengths of the fluorescence detector were set, respectively, to 460 and 520 nm. FSEC chromatograms were plotted using Plot2 software and data was normalized.