Vivaspin 500 centrifugal concentrator

Cell pellets corresponding to 200 mL culture were resuspended in 4 mL of breaking buffer (50 mM sodium phosphate buffer, pH 7.5, 1 mM EDTA, 5% (v/v) glycerol, 10 mM imidazole, 1 mM PMSF) to obtain an OD₆₀₀ of 50. Lysis of cells was achieved by addition of glass beads and repeated vortexing for 30 s followed by 30 s incubation on ice. After 20 min of treatment cell debris was removed by centrifugation (14.800g, 4 °C, 10 min). Protein crude extract was loaded on a gravity column with 1 mL Profinity™ IMAC Ni-charged Resin (Bio-Rad Laboratories, München, Germany) and incubated for 2 h with orbital shaking at 4 °C. After washing with 10 mL of 50 mM sodium phosphate buffer (pH 7.5) containing 300 mM NaCl and 30 mM imidazole, elution was performed with 600 µL of 50 mM sodium phosphate buffer (pH 7.5) containing 300 mM NaCl and 500 mM imidazole. The elution was repeated three more times. Fractions with LOX activity were pooled, dialyzed overnight against 50 mM sodium phosphate buffer (pH 7.0) and concentrated by ultrafiltration using Vivaspin 500 centrifugal concentrators (Sartorius Stedim Biotech, Göttingen, Germany) with a molecular weight cutoff of 50 000. Samples were analyzed via SDS–PAGE and protein concentration was determined by the Bradford method using bovine serum albumin as standard.^{33 (link)} Protein was stored in small aliquots at −80 °C until required.