Figure 2

SDS-PAGE analysis of rhANXA5 purification steps. SDS-PAGE (12%) analysis of fractions from different steps of rhANXA5 purification. Each sample lane contains 8 μg of total protein. M corresponds to Thermo Scientific™ Unstained Protein MW Marker; lane 1: cells suspended in buffer A (50 mM Tris–HCl, pH 7.2, 10 mM CaCl₂) with no previous centrifugation step; lane 2: supernatant of cells in buffer A after sonication and centrifugation (for 20 min, 24,400 × g, 4°C); lane 3: cell pellet from the previous centrifugation suspended in buffer B (50 mM Tris–HCl, pH 7.2, 20 mM EDTA); lane 4: suspension from previous step dialyzed against 2 L of 20 mM Tris–HCl, pH 8.0 for 3 times, overnight in the first step, followed by two steps of dialysis 2 h each; lane 5: supernatant after centrifugation of dialyzed sample (for 30 min, 24,400 × g, 4°C); lane 6: homogeneous preparation of annexin V eluted from the Mono Q column.