Miniature Gelatin Affinity Chromatography for Gelatinase Purification

Gelatinase purification was performed by miniature gelatin affinity chromatography [19 (link)]. Briefly, 0.5 mL of pooled sera of five lung cancer patients, diluted with 0.5 mL of 50 mM Tris pH 7.5, 0.6 M NaCl (equilibration buffer), were added to mini-spin columns (Bio-Rad Laboratories, Segrate, MI, Italy) loaded with 0.5 mL gelatin-Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) previously equilibrated with equilibration buffer. The mixture was then equilibrated at 4 °C for 60 min (vortexing every 10 min and reversing the column). Successively, the cap was removed and the unbound sample was removed by centrifugation at 3000× g (Amicon MC-13 microcentrifuge; Millipore, Bedford, MA, USA), for 2 min. Beads were washed two times with washing buffer 50 mM Tris pH 7.5, 0.1 M NaCl, and gelatinases were eluted with 150 μL of 5% DMSO in 50 mM Tris pH 7.5, 0.1 M NaCl after incubation for 30 min. at room temperature. The eluate was desalted and concentrated to a final volume of 30 μL with 2.0 mL of water in Vivaspin 500 (MWCO 30,000; GE Healthcare, Uppsala, Sweden). A total of 5 µL of purified gelatinase was applied for 2-DZ analysis, whereas 25 µL of sample was subjected to 2-DE analysis for western blot identification of 65 kDa MMP-9 active form.