Drugs have been separated on a C18 column using an elution gradient based on an increase in the acetonitrile percentage.
Human serum (0.5 ml) was fractionated on a 24 ml Sephacryl 200-HR column using an elution buffer consisting of 150 mM NaCl and 10 mM tris pH 7.4 and a flow rate of 5.0 ml/hr.
The third step is the actual chromatographic separation on the analytical LC column using an elution gradient.
The protein was eluted with a buffer containing 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole and further purified by size exclusion chromatography on a HiLoad Superdex 75 column (GE Healthcare, USA) using an elution buffer containing 20 mM Tris-HCl pH 7.5, and 150 mM NaCl.
Reactions were subjected to 95° for 10 min, followed by 30 sec at 95°, 30 sec at 64°, 3 min at 72°, with repetition of steps 2 4 for 11 cycles, followed by 7 min at 72° and then held at 4°. Amplicons were then purified using a Qiagen Qiaquick column with elution in 50 µl of water.
Analysis of sugar monomers in all hydrolysates was determined by high-performance anion exchange chromatography with pulsed amperometric detection (Dionex, UK), using a CarboPac PA20 column with isocratic elution at a working flow rate of 0.5 ml/min.
The reactions were stopped by adding 0.1 M disodium edetate (EDTA) and the radiolabelled DARPins were purified by elution into PBS using a NAP-5 column (GE Healthcare, Little Chalfont, UK) pre-equilibrated with PBS.
IMAC of serum free NS0 samples was performed on a 20 ml C16/20 Chelating Sepharose Fast Flow column (GE Health), under the same buffer conditions as the 1 ml Hitrap columns, but using a gradient elution with 20 mM PP buffer containing 500 mM imidazole and 0.5 M NaCl.
His-tagged proteins were purified to ∼99% purity from bacterial lysates using a Histrap HP column (GE Healthcare) and a gradient elution profile according to the manufacturer's protocol.
The SEC analysis was performed using a Superdex 200 column equipped on an ÄKTA FPLC with an elution rate of 0.5 mL/min.
