Tsk gel g3000swxl 7.8 300 mm column

Analytical size-exclusion (SE) was performed using a Dionex HPLC instrument (Pump P580, Auto sampler ASI-100/ASI-100 T Injector, UV/VIS Detector UVD340U, Chromeleon 6.80 Software) with Tosoh Bioscience TSK-Gel G3000SWXL 7.8×300 mm column. Phosphate Buffered Saline (PBS) was used as the mobile phase and samples of <50 μg or 100 μg were injected.
Reference standards and 25% Gel Filtration Standard (GFS, BioRad) were run before and after the samples. The separation was performed using an isocratic separation method with a runtime of 20 min and a flow rate of 1 ml/min. The column oven was set at 25°C and the sample holder at 8°C. The size distribution profiles were recorded using UV absorption at 214 nm.

20 µg purified protein sample (100 μL of 0.2 mg/mL stock diluted in PBS, pH7.4) was injected onto either a Superdex 200 10/300 GL Tricorn column (GE Healthcare, Little Chalfont, UK) or a TSK Gel G3000SWXL, 7.8 × 300 mm, column (Tosoh Bioscience, Reading, UK) 3 days post-purification and developed respectively with an isocratic gradient of PBS, pH 7.4 at 1 mL/min or 200 mM phosphate, pH 7.0 at 1 mL/min. Signal detection was by absorbance at 280 nm. Gel filtration protein standards (BioRad, Watford, UK) were loaded for molecular weight estimation (chromatograms are shown in Supplementary Figure S1). Since all purified fusion proteins contain disulphide stabilised Fv/scFv, the observed monomer level is independent of protein concentration and unaffected by on-column dilution effects.

SEC was carried out with a TSKgel G3000 SWXL, 7.8 × 300 mm column (Tosoh) connected to a HPLC system (Tosoh). The isocratic elution was carried out with a mobile phase 0.1 M sodium phosphate, 0.1 M sodium sulphate, 0.05% sodium azide pH 6.7 at a flow rate of 1.0 mL/min. The column temperature was kept at 30 °C during analysis. Fifty µL of each 200 µg/mL sample was injected onto the column and analysed with UV detection at the wavelength of 280 nm.