Nuvia imac ni charged resin

Histidine (His)-tagged recombinant mZP2-3 and mIzumo1-3 were partially purified via immobilized metal affinity chromatography (IMAC) using a Ni-chelating resin. Leaf extracts (in 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM sucrose, 5 mM imidazole) were centrifuged at 16,000 × g and 4°C for 1 h. Supernatants were collected and loaded onto a column (Bio-Rad Laboratories, Hercules, California, USA) containing pre-equilibrated Nuvia IMAC Ni-charged resin (Bio-Rad Laboratories). The column was washed twice with the wash buffer containing 20 and 30 mM imidazole. Recombinant protein was eluted with an elution buffer containing 300 mM imidazole, and the elution fraction was concentrated and desalted using Vivaspin 20 centrifugal concentrators with a 10 kDa cut-off membrane (Sartorius AG, Germany). LC-MS analyses of partially purified mIzumo1-3 protein were performed as already described (Klähn et al., 2021 (link)).

His-tagged recombinant mZP3-1, GFP-mZP3-1, and mZP3-3 were purified using immobilized metal affinity chromatography (IMAC) based on the affinity for the 6His purification tag. For large-scale protein extraction, 30 g of ground leaf material was homogenized in ice-cooled protein extraction buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM sucrose, 5 mM imidazole) using a Homogenizer (Polytron^® PT 2100, Kinematica AG, Malters, Switzerland) for 30 s at 30,000 rpm. Lysates were centrifuged at 4600× g for 15 min and then clarified by repeated ultracentrifugation at 16,000× g for 30 min at 4 °C. The supernatants were collected and loaded onto a column (Bio-Rad Laboratories, Hercules, CA, USA) containing pre-equilibrated Nuvia™ IMAC Ni-charged resin (Bio-Rad Laboratories, Hercules, CA, USA). The column was washed with washing buffer (50 mM NaH₂PO₄, 20 mM imidazole, 20 mM NaCl, pH 8). The target recombinant protein was further eluted with elution buffer (50 mM NaH₂PO₄, 300 mM imidazole, 300 mM NaCl, pH 8). The elution fraction was concentrated and desalted using Vivaspin 20 centrifugal concentrators with a 10 kDa cut-off membrane (Sartorius AG, Göttingen, Germany).