Recombinant proteins were analyzed by 13% SDS-PAGE under reducing conditions. The separated proteins were transferred onto a 0.45-μm PVDF membrane (Millipore) in semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). The PVDF membrane (Cytiva, Marlborough, MA) containing recombinant protein was blocked with 5% skim milk in PBS-T (0.5% v/v Tween-20 in 1× PBS) and then incubated with anti-GST antibody (Novagen, Reno, NV) and mouse and rabbit immune sera diluted 1:2,000 in PBS-T. After the primary antibody reaction, the membrane was incubated with the secondary IRDye® goat anti-mouse (1: 5,000 dilution) or IRDye® goat anti-rabbit (1: 5,000) (Li-COR® Bioscience, Lincoln, NE) antibodies to detect antigens. The results were visualized in the Odyssey infrared imaging system and analyzed with Odyssey software (Li-COR® Bioscience) (Lee et al., 2016 (link)).
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