Kta avant 25 fplc system

Recombinant proteins (AfKatG, MDBP, Taq, MutTaq) were expressed in fusion with His-tag sequence and afterwards purified using affinity chromatography on ÄKTA Avant 25 FPLC system (GE Healthcare) with 1 ml HisTrap HP (GE Healthcare). At the beginning of the purification run, the column was washed with 5 column volumes (5 CV) of equilibration buffer (50 mM TrisHCl, pH 8.0, 0.5 M NaCl). Sample was loaded onto column using the pump at speed of 0.1-1 CV/min. Afterwards, the column was washed with equilibration buffer (10 CV) with 5% addition of elution buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 0.5 M imidazole) to remove non-bound and non-specifically bound proteins. Elution buffer with 0.5 M imidazole was used to release bound His-tagged proteins. Samples were taken automatically by the fraction collector based on predefined parameters.

Recombinant proteins (AfKatG, MDBP, Taq, MutTaq) were expressed in fusion with His-tag sequence and afterwards purified using affinity chromatography on ÄKTA Avant 25 FPLC system (GE Healthcare) with 1 ml HisTrap HP (GE Healthcare). At the beginning of the purification run, the column was washed with 5 column volumes (5 CV) of equilibration buffer (50 mM TrisHCl, pH 8.0, 0.5 M NaCl). Sample was loaded onto column using the pump at speed of 0.1-1 CV/min. Afterwards, the column was washed with equilibration buffer (10 CV) with 5% addition of elution buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 0.5 M imidazole) to remove non-bound and non-specifically bound proteins. Elution buffer with 0.5 M imidazole was used to release bound His-tagged proteins. Samples were taken automatically by the fraction collector based on predefined parameters.

The chromatopanning called biopanning procedure described here was modified from Schönberger et al., 2019 [14 (link)] and first published by Nian et al., 2010 [15 (link)]. Target material were arsenic oxyanions, arsenous acid and arsenous anions (H₃AsO₃⁻, H₂AsO₄⁻, HAsO₄²⁻, AsO₄³⁻) of trivalent As(III) and pentavalent As(V) immobilized on a monolithic ion exchange column (CIM^® QA Disk Monolithic Column, BIA Separations, Ajdovščina, Slovenia) in a chromatographic setup using an Äkta avant 25 FPLC system (GE Healthcare, Amersham, UK).
In this study, phage were incubated with the unloaded column in a pre-screening (negative biopanning) for removal of unspecific binding phage followed by enrichment of binding phage in three rounds of positive chromatopanning against the immobilized target material.