Initially, the protein extract solution was subject to ion exchange column chromatography using SP Sepharose Fast Flow (GE Healthcare, Sweden). The column (with 5 ml of stationary phase) was equilibrated with five column volumes of sodium acetate/acetic acid 10mM pH5 and 10 ml of protein extract containing bacteriocin were injected into this. After washing the column with sodium acetate/acetic acid 10 mM pH5 (Buffer A), elution was performed using a linear gradient from sodium acetate/acetic acid 10mM pH5 in 0 minutes to 100% of NaCl 1M in sodium acetate/acetic acid 10mM pH5 (Buffer B) in 25 minutes in ÄKTA pure chromatography system (GE Healthcare), with a flow rate 1 ml/min. Protein and peptides absorbance were detected at 280 nm. 2 ml fractions were collected automatically and tested for antimicrobial activity as previously described in point
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So, afterward column equilibration with five column volumes of sodium acetate/acetic acid 10mM pH5, 10 ml of protein extract containing bacteriocin were injected into this. The column was washed with five column volumes of sodium acetate/acetic acid 10mM pH5 and the elution of bacteriocin performed by increasing steps of NaCl concentration (0.3M, 0.6M and 1.0M of NaCl in sodium acetate/acetic acid 10mM pH5 buffer). Similar to linear elution, the stepwise elution strategy was performed in ÄKTA pure chromatography system (GE Healthcare) with a flow rate 1 ml/min. Protein and peptides absorbance were detected at 280 nm. 2 ml fractions were collected automatically and tested for antimicrobial activity as previously described in point Bacteriocin detection and activity assay. After checking optimal step for bacteriocin elution (designed as Fraction I), only this was collected for subsequent purification and activity
So, afterward column equilibration with five column volumes of sodium acetate/acetic acid 10mM pH5, 10 ml of protein extract containing bacteriocin were injected into this. The column was washed with five column volumes of sodium acetate/acetic acid 10mM pH5 and the elution of bacteriocin performed by increasing steps of NaCl concentration (0.3M, 0.6M and 1.0M of NaCl in sodium acetate/acetic acid 10mM pH5 buffer). Similar to linear elution, the stepwise elution strategy was performed in ÄKTA pure chromatography system (GE Healthcare) with a flow rate 1 ml/min. Protein and peptides absorbance were detected at 280 nm. 2 ml fractions were collected automatically and tested for antimicrobial activity as previously described in point Bacteriocin detection and activity assay. After checking optimal step for bacteriocin elution (designed as Fraction I), only this was collected for subsequent purification and activity