Efficient Purification of PMab-2 Antibody

PMab-2 was purified following a previously described method (Ocampo et al., 2016 (link)) with some modifications. Briefly, 10 g of agroinfiltrated N. benthamiana leaves were ground in liquid nitrogen using a mortar and pestle. The powdered tissue was mixed with 40 ml of extraction buffer (0.5 NaCl, 45 mM Tris-HCl, 1 mM EDTA, 40 mM ascorbic acid, 1 mM PMSF; pH 7.5) and then agitated on ice for 1 h. The suspension was filtered through Miracloth (Meck Millipore), and centrifuged twice at 44,000 ×g for 30 min at 4°C. The supernatant was filtered through an Omnipore Membrane Filter (pore size 0.2 µm; Merck Millipore). PMab-2 was then purified using the AKTA start system equipped with a HiTrap Protein G HP column (GE Healthcare). Once the antibodies had bound to Protein G, the column was washed with the aforementioned extraction buffer without ascorbic acid. The antibodies were eluted with 0.1 M glycine-HCl (pH 2.7) and neutralized with 60 µl of 1 M Tris-HCl (pH 9.0) per ml fraction. The elutant was concentrated using Vivaspin (Sartorius) with 1 × PBS, at approximately 20 times.